Henipavirus vaccine

ABSTRACT

The present invention is directed to an artificial nucleic acid and to polypeptides suitable for use in treatment or prophylaxis of an infection with Henipavirus, particularly Hendra virus and/or Nipah virus or a disorder related to such an infection. In particular, the present invention concerns a Hendra virus and/or Nipah virus vaccine. The present invention is directed to an artificial nucleic acid, polypeptides, compositions and vaccines comprising the artificial nucleic acid or the polypeptides. The invention further concerns a method of treating or preventing a disorder or a disease, first and second medical uses of the artificial nucleic acid, polypeptides, compositions and vaccines. Further, the invention is directed to a kit, particularly to a kit of parts, comprising the artificial nucleic acid, polypeptides, compositions and vaccines.

This application is a national phase application under 35 U.S.C. § 371of International Application No. PCT/EP2017/084525, filed Dec. 22, 2017,which claims benefit of International Application No. PCT/EP2016/082672,filed Dec. 23, 2016, the entire contents of each of which are herebyincorporated by reference.

The present invention is directed to an artificial nucleic acid and topolypeptides suitable for use in treatment or prophylaxis of aninfection with Hendra virus and/or Nipah virus or a disorder related tosuch an infection. In particular, the present invention concerns aHendra virus and/or Nipah virus vaccine. The present invention isdirected to an artificial nucleic acid, polypeptides, compositions andvaccines comprising the artificial nucleic acid or the polypeptides. Theinvention further concerns a method of treating or preventing a disorderor a disease associated with a Hendra virus and/or Nipah virusinfection, first and second medical uses of the artificial nucleic acid,polypeptides, compositions and vaccines. Further, the invention isdirected to a kit, particularly to a kit of parts, comprising theartificial nucleic acid, polypeptides, compositions and vaccines.

Henipavirus is a genus of negative sense single stranded RNA virusesbelonging to the Paramyxovirinae virus superfamily. The Henipavirusgenome is about 18 kb in size, encoding for nine proteins, comprisingRNA-directed RNA polymerase (L), fusion protein (F), non-structuralprotein (V), glycoprotein (G), nucleoprotein (N), matrix protein (M),phosphoprotein (P), protein C, and protein W. The genus currentlycontains five described and established species, including thepathogenic viruses Hendra virus and Nipah virus.

Hendra virus is the source of a recently emerging disease in animals andhuman. Hendra virus was first recognized in September 1994 after anoutbreak of respiratory illness among twenty horses and two humans inHendra, Queensland, Australia. In 1995, a second unrelated outbreak wasidentified that had occurred in August 1994 in Mackay, Queensland, inwhich two horses died and one human became. Four of the seven people whocontracted the virus from infected horses have died since the diseasefirst emerged in 1994. The fatality rate has been reported at more than70% in horses and 50% in humans.

The Nipah virus was initially isolated in 1999 upon examining samplesfrom an outbreak of encephalitis and respiratory illness among adult menin Malaysia and Singapore. The host for Nipah virus is still unknown,but flying foxes (bats of the Pteropus genus) are suspected to be thenatural host. Infection with Nipah virus in humans has been associatedwith encephalitis characterized by fever and drowsiness and more seriouscentral nerve system disease, such as coma, seizures and inability tomaintain breathing. Illness with Nipah virus begins with 3-14 days offever and headache, followed by drowsiness and disorientationcharacterized by mental confusion. These signs and symptoms can progressto coma within 24-48 hours. Some patients have had a respiratory illnessduring the early part of their infections. Serious nerve disease withNipah virus encephalitis has been marked by some sequelae, such aspersistent convulsions and personality changes. During a Nipah virusdisease outbreak in 1998-1999, about 40% of the patients with seriousnerve disease who entered hospitals died from the illness.

Hendra virus and Nipah virus, like the majority of otherparamyxoviruses, possess two surface glycoproteins, a fusion protein (F)and a glycoprotein protein (G), both involved in promotion of fusionbetween the viral membrane and the membrane of the target host cell.Hendra viruses and Nipah viruses require both their attachment andfusion proteins to initiate membrane fusion. Various studies wereconducted to understand the functions of the G and F proteins in virusinfection.

Current vaccine approaches for protection from Nipah virus infectionhave focused on the use of Nipah virus glycoprotein (G) and/or fusionprotein (F) as immunogens in various platforms, including DNA vaccines,subunit vaccines, non-replicating vectors, as well as replicatingvectors.

To date, no effective antiviral therapies have been approved for eitherthe prevention or treatment of diseases caused by Hendra virusinfections and/or Nipah virus infections. Thus, there is a significantunmet medical need to find agents that can prevent Hendra virus and/orNipah virus infection, shorten the duration of Hendra virus and/or Nipahvirus-induced illness, lessen the severity of symptoms, minimizesecondary bacterial infections and exacerbations of underlying disease,and reduce virus transmission. A prophylactic Hendra virus and Nipahvirus vaccine should be protective against a wide variety of serotypesto reduce the number of Hendra virus and Nipah virus infections, hence,reducing the risk of a global pandemic threat.

The underlying object of the present invention is therefore to provide aHendra virus and/or Nipah virus vaccine. It is a further preferredobject of the invention to provide a Hendra virus and/or Nipah virusvaccine, which may be produced in a fast manner at an industrial scalein a potential pandemic scenario. A further object of the presentinvention is the provision of a storage-stable Hendra virus and/or Nipahvirus vaccine. Further object of the underlying invention is to providenucleic acid sequences, particularly mRNA sequences coding for antigenicpeptides or proteins derived from a protein of a Hendra virus and/orNipah virus or a fragment or variant thereof for the use as a vaccinefor prophylaxis or treatment of Hendra virus and/or Nipah virusinfections. Furthermore, it is the object of the present invention toprovide an effective Hendra virus and/or Nipah virus vaccine which canbe stored without cold chain and which enables rapid and scalablevaccine production which is of major importance in the context ofpandemic Hendra virus and/or Nipah virus outbreaks.

The object underlying the present invention is solved by the claimedsubject-matter.

Definitions

For the sake of clarity and readability the following definitions areprovided. Any technical feature mentioned for these definitions may beread on each and every embodiment of the invention. Additionaldefinitions and explanations may be specifically provided in the contextof these embodiments.

Adaptive immune response: The adaptive immune response is typicallyunderstood to be an antigen-specific response of the immune system.Antigen specificity allows for the generation of responses that aretailored to specific pathogens or pathogen-infected cells. The abilityto mount these tailored responses is usually maintained in the body by“memory cells”. Should a pathogen infect the body more than once, thesespecific memory cells are used to quickly eliminate it. In this context,the first step of an adaptive immune response is the activation of naïveantigen-specific T cells or different immune cells able to induce anantigen-specific immune response by antigen-presenting cells. Thisoccurs in the lymphoid tissues and organs through which naïve T cellsare constantly passing. The three cell types that may serve asantigen-presenting cells are dendritic cells, macrophages, and B cells.Each of these cells has a distinct function in eliciting immuneresponses. Dendritic cells may take up antigens by phagocytosis andmacropinocytosis and may become stimulated by contact with e.g. aforeign antigen to migrate to the local lymphoid tissue, where theydifferentiate into mature dendritic cells. Macrophages ingestparticulate antigens such as bacteria and are induced by infectiousagents or other appropriate stimuli to express MHC molecules. The uniqueability of B cells to bind and internalize soluble protein antigens viatheir receptors may also be important to induce T cells. MHC-moleculesare, typically, responsible for presentation of an antigen to T-cells.Therein, presenting the antigen on MHC molecules leads to activation ofT cells which induces their proliferation and differentiation into armedeffector T cells. The most important function of effector T cells is thekilling of infected cells by CD8+ cytotoxic T cells and the activationof macrophages by Th1 cells which together make up cell-mediatedimmunity, and the activation of B cells by both Th2 and Th1 cells toproduce different classes of antibody, thus driving the humoral immuneresponse. T cells recognize an antigen by their T cell receptors whichdo not recognize and bind the antigen directly, but instead recognizeshort peptide fragments e.g. of pathogen-derived protein antigens, e.g.so-called epitopes, which are bound to MHC molecules on the surfaces ofother cells.

Adaptive immune system: The adaptive immune system is essentiallydedicated to eliminate or prevent pathogenic growth. It typicallyregulates the adaptive immune response by providing the vertebrateimmune system with the ability to recognize and remember specificpathogens (to generate immunity), and to mount stronger attacks eachtime the pathogen is encountered. The system is highly adaptable becauseof somatic hyper mutation (a process of accelerated somatic mutations),and V(D)J recombination (an irreversible genetic recombination ofantigen receptor gene segments). This mechanism allows a small number ofgenes to generate a vast number of different antigen receptors, whichare then uniquely expressed on each individual lymphocyte. Because thegene rearrangement leads to an irreversible change in the DNA of eachcell, all of the progeny (offspring) of such a cell will then inheritgenes encoding the same receptor specificity, including the Memory Bcells and Memory T cells that are the keys to long-lived specificimmunity.

Adjuvant/adjuvant component: An adjuvant or an adjuvant component in thebroadest sense is typically a pharmacological and/or immunological agentthat may modify, e.g. enhance, the effect of other agents, such as adrug or vaccine. It is to be interpreted in a broad sense and refers toa broad spectrum of substances. Typically, these substances are able toincrease the immunogenicity of antigens. For example, adjuvants may berecognized by the innate immune systems and, e.g., may elicit an innateimmune response. “Adjuvants” typically do not elicit an adaptive immuneresponse. Insofar, “adjuvants” do not qualify as antigens. Their mode ofaction is distinct from the effects triggered by antigens resulting inan adaptive immune response.

Antigen: In the context of the present invention “antigen” referstypically to a substance which may be recognized by the immune system,preferably by the adaptive immune system, and is capable of triggeringan antigen-specific immune response, e.g. by formation of antibodiesand/or antigen-specific T cells as part of an adaptive immune response.Typically, an antigen may be or may comprise a peptide or protein whichmay be presented by the MHC to T-cells. In the sense of the presentinvention an antigen may be the product of translation of a providednucleic acid molecule, preferably an mRNA as defined herein. In thiscontext, also fragments, variants and derivatives of peptides andproteins comprising at least one epitope are understood as antigens.

Antigenic peptide or protein: An antigenic peptide or protein is apeptide or protein derived from a protein which may stimulate the body'sadaptive immune system to provide an adaptive immune response. Thereforean antigenic peptide or protein comprises at least one epitope of theprotein it is derived from.

Artificial nucleic acid molecule: The terms “artificial nucleic acidmolecule” and “artificial nucleic acid” may typically be understood tobe a nucleic acid molecule, e.g. a DNA or an RNA that does not occurnaturally. In other words, an artificial nucleic acid molecule may beunderstood as a non-natural nucleic acid molecule. Such nucleic acidmolecule may be non-natural due to its individual sequence (which doesnot occur naturally) and/or due to other modifications, e.g. structuralmodifications of nucleotides which do not occur naturally. An artificialnucleic acid molecule may be a DNA molecule, an RNA molecule or ahybrid-molecule comprising DNA and RNA portions. Typically, artificialnucleic acid molecules may be designed and/or generated by geneticengineering methods to correspond to a desired artificial sequence ofnucleotides (heterologous sequence). In this context an artificialsequence is usually a sequence that may not occur naturally, i.e. itdiffers from the wild type sequence by at least one nucleotide. The term“wild type” may be understood as a sequence occurring in nature.Further, the term “artificial nucleic acid molecule” is not restrictedto mean “one single molecule” but is, typically, understood to comprisean ensemble of identical molecules. Accordingly, it may relate to aplurality of identical molecules contained in an aliquot.

Bicistronic nucleic acid, multicistronic nucleic acid: A bicistronic ormulticistronic nucleic acid is typically an RNA or DNA, preferably anmRNA that typically may have two (bicistronic) or more (multicistronic)coding sequences. A coding sequence in this context is a sequence ofcodons that is translatable into a peptide or protein.

Carrier/polymeric carrier: A carrier in the context of the invention maytypically be a compound that facilitates transport and/or complexationof another compound (cargo). A polymeric carrier is typically a carrierthat is formed of a polymer. A carrier may be associated to its cargo bycovalent or non-covalent interaction. A carrier may transport nucleicacids, e.g. RNA or DNA, to the target cells. The carrier may—for someembodiments—be a cationic component.

Cationic component: The term “cationic component” or “cationic compound”typically refers to a charged molecule, which is positively charged(cation) at a pH value typically from 1 to 9, preferably at a pH valueof or below 9 (e.g. from 5 to 9), of or below 8 (e.g. from 5 to 8), ofor below 7 (e.g. from 5 to 7), most preferably at a physiological pH,e.g. from 7.3 to 7.4. Accordingly, a cationic component, e.g. a cationicpeptide, cationic polysaccharide, a cationic lipid may be any positivelycharged compound or polymer, preferably a cationic peptide or proteinwhich is positively charged under physiological conditions, particularlyunder physiological conditions in vivo. A “cationic peptide or protein”may contain at least one positively charged amino acid, or more than onepositively charged amino acid, e.g. selected from Arg, His, Lys or Orn.Accordingly, “polycationic” components are also within the scopeexhibiting more than one positive charge under the conditions given.

Cap analogue: A cap analogue refers to a non-polymerizable di-nucleotidethat has cap functionality in that it facilitates translation orlocalization, and/or prevents degradation of a nucleic acid molecule,particularly of an RNA molecule, when incorporated at the 5′-end of thenucleic acid molecule. Non-polymerizable means that the cap analoguewill be incorporated only at the 5′ terminus because it does not have a5′ triphosphate and therefore cannot be extended in the 3′ direction bya template-dependent polymerase, particularly, by template-dependent RNApolymerase. Cap analogues include, but are not limited to, a chemicalstructure selected from the group consisting of m7GpppG, m7GpppA,m7GpppC; unmethylated cap analogues (e.g., GpppG); dimethylated capanalogue (e.g., m2,7GpppG), trimethylated cap analogue (e.g.,m2,2,7GpppG), dimethylated symmetrical cap analogues (e.g., m7Gpppm7G),or anti reverse cap analogues (e.g., ARCA; m7,2′OmeGpppG, m7,2′dGpppG,m7,3′OmeGpppG, m7,3′dGpppG and their tetraphosphate derivatives)(Stepinski et al., 2001. RNA 7(10):1486-95). Further cap analogues havebeen described previously (U.S. Pat. No. 7,074,596, WO 2008/016473, WO2008/157688, WO 2009/149253, WO 2011/015347, and WO 2013/059475). Thesynthesis of N7-(4-chlorophenoxyethyl) substituted dinucleotide capanalogues has been described recently (Kore et al. (2013) Bioorg. Med.Chem. 21(15): 4570-4).

5′-cap-Structure: A 5′-cap is typically a modified nucleotide (capanalogue), particularly a guanine nucleotide, added to the 5′-end of anucleic acid molecule, particularly of an RNA molecule, e.g. an mRNAmolecule. Preferably, the 5′-cap is added using a 5′-5′-triphosphatelinkage (also named m7GpppN). Further examples of 5′-cap structuresinclude glyceryl, inverted deoxy abasic residue (moiety), 4′,5′methylene nucleotide, 1-(beta-D-erythrofuranosyl) nucleotide, 4 ‘-thionucleotide, carbocyclic nucleotide, 1,5-anhydrohexitol nucleotide,L-nucleotides, alpha-nucleotide, modified base nucleotide,threo-pentofuranosyl nucleotide, acyclic 3’,4″-seco nucleotide, acyclic3,4-dihydroxybutyl nucleotide, acyclic 3,5 dihydroxypentyl nucleotide,3′-3′-inverted nucleotide moiety, 3′-3′-inverted abasic moiety,3′-2′-inverted nucleotide moiety, 3′-2′-inverted abasic moiety,1,4-butanediol phosphate, 3′-phosphoramidate, hexylphosphate, aminohexylphosphate, 3′-phosphate, 3′ phosphorothioate, phosphorodithioate, orbridging or non-bridging methylphosphonate moiety. These modified 5′-capstructures may be used in the context of the present invention to modifythe mRNA sequence of the inventive composition. Further modified 5′-capstructures which may be used in the context of the present invention arecap 1 (additional methylation of the ribose of the adjacent nucleotideof m7GpppN), cap2 (additional methylation of the ribose of the 2ndnucleotide downstream of the m7GpppN), cap3 (additional methylation ofthe ribose of the 3rd nucleotide downstream of the m7GpppN), cap4(additional methylation of the ribose of the 4th nucleotide downstreamof the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g.phosphothioate modified ARCA), inosine, N1-methyl-guanosine,2′-fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine,2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In the contextof the present invention, a 5′-cap (cap0 or cap1) structure may also beformed in chemical RNA synthesis or RNA in vitro transcription(co-transcriptional capping) using cap analogues, or a cap structure maybe formed in vitro using capping enzymes (e.g., commercially availablecapping kits) or using immobilized capping enzymes, e.g. in a cappingreactor (WO 2016/193226).

Cellular immunity/cellular immune response: Cellular immunity relatestypically to the activation of macrophages, natural killer cells (NK),antigen-specific cytotoxic T-lymphocytes, and the release of variouscytokines in response to an antigen. In more general terms, cellularimmunity is not based on antibodies, but on the activation of cells ofthe immune system. Typically, a cellular immune response may becharacterized e.g. by activating antigen-specific cytotoxicT-lymphocytes that are able to induce apoptosis in cells, e.g. specificimmune cells like dendritic cells or other cells, displaying epitopes offoreign antigens on their surface. Such cells may be virus-infected orinfected with intracellular bacteria, or cancer cells displaying tumorantigens. Further characteristics may be activation of macrophages andnatural killer cells, enabling them to destroy pathogens and stimulationof cells to secrete a variety of cytokines that influence the functionof other cells involved in adaptive immune responses and innate immuneresponses.

Chemical synthesis of nucleic acids: Chemical synthesis of relativelyshort fragments of oligonucleotides with defined chemical structureprovides a rapid and inexpensive access to custom-made oligonucleotidesof any desired sequence. Whereas enzymes synthesize DNA and RNA only inthe 5′ to 3′ direction, chemical oligonucleotide synthesis does not havethis limitation, although it is most often carried out in the opposite,i.e. the 3′ to 5′ direction. Currently, the process is implemented assolid-phase synthesis using the phosphoramidite method andphosphoramidite building blocks derived from protected nucleosides (A,C, G, and U), or chemically modified nucleosides. To obtain the desiredoligonucleotide, the building blocks are sequentially coupled to thegrowing oligonucleotide chain on a solid phase in the order required bythe sequence of the product in a fully automated process. Upon thecompletion of the chain assembly, the product is released from the solidphase to the solution, deprotected, and collected. The occurrence ofside reactions sets practical limits for the length of syntheticoligonucleotides (up to about 200 nucleotide residues), because thenumber of errors increases with the length of the oligonucleotide beingsynthesized. Products are often isolated by HPLC to obtain the desiredoligonucleotides in high purity. Chemically synthesized oligonucleotidesfind a variety of applications in molecular biology and medicine. Theyare most commonly used as antisense oligonucleotides, small interferingRNA, primers for DNA sequencing and amplification, probes for detectingcomplementary DNA or RNA via molecular hybridization, tools for thetargeted introduction of mutations and restriction sites, and for thesynthesis of artificial genes. Moreover, long-chain DNA molecules andlong-chain RNA molecules may be chemically synthetized and used in thecontext of the present invention.

Cloning site: A cloning site is typically understood to be a segment ofa nucleic acid molecule, which is suitable for insertion of a nucleicacid sequence, e.g., a nucleic acid sequence comprising a codingsequence. Insertion may be performed by any molecular biological methodknown to the one skilled in the art, e.g. by restriction and ligation. Acloning site typically comprises one or more restriction enzymerecognition sites (restriction sites). These one or more restrictionssites may be recognized by restriction enzymes which cleave the DNA atthese sites. A cloning site which comprises more than one restrictionsite may also be termed a multiple cloning site (MCS) or a polylinker.

Coding sequence: A coding sequence (cds) in the context of the inventionis typically a sequence of several nucleotide triplets, which may betranslated into a peptide or protein. A coding sequence preferablycontains a start codon, i.e. a combination of three subsequentnucleotides coding usually for the amino acid methionine (ATG), at its5′-end and a subsequent region which usually exhibits a length which isa multiple of 3 nucleotides. A coding sequence is preferably terminatedby a stop-codon (e.g., TAA, TAG, and TGA). Typically, this is the onlystop-codon of the coding sequence. Thus, a coding sequence in thecontext of the present invention is preferably a nucleotide sequence,consisting of a number of nucleotides that may be divided by three,which starts with a start codon (e.g. ATG) and which preferablyterminates with a stop codon (e.g., TAA, TGA, or TAG). The codingsequence may be isolated or it may be incorporated in a longer nucleicacid sequence, for example in a vector or an mRNA. In the context of thepresent invention, a coding sequence may also be termed “protein codingregion”, “coding sequence”, “cds”, “open reading frame” or “ORF”.

Derived from: The phrase “derived from” as used throughout the presentspecification in the context of a nucleic acid, i.e. for a nucleic acid“derived from” (another) nucleic acid, means that the nucleic acid,which is derived from (another) nucleic acid, shares at least 50%,preferably at least 55%, preferably at least 60%, preferably at least65%, preferably at least 70%, more preferably at least 75%, morepreferably at least 80%, 81%, 82%, 83%, 84%, more preferably at least85%, 86%, 87%, 88%, 89% even more preferably at least 90%, 91%, 92%,93%, 94%, even more preferably at least 95%, 96%, 97%, and particularlypreferably at least 98%, 99% sequence identity with the nucleic acidfrom which it is derived. The skilled person is aware that sequenceidentity is typically calculated for the same types of nucleic acids,i.e. for DNA sequences or for RNA sequences. Thus, it is understood, ifa DNA is “derived from” an RNA or if an RNA is “derived from” a DNA, ina first step the RNA sequence is converted into the corresponding DNAsequence (in particular by replacing the uracils (U) by thymidines (T)throughout the sequence) or, vice versa, the DNA sequence is convertedinto the corresponding RNA sequence (in particular by replacing thethymidines (T) by uracils (U) throughout the sequence). Thereafter, thesequence identity of the DNA sequences or the sequence identity of theRNA sequences is determined. Preferably, a nucleic acid “derived from” anucleic acid also refers to nucleic acid, which is modified incomparison to the nucleic acid from which it is derived, e.g. in orderto increase RNA stability even further and/or to prolong and/or increaseprotein production. It goes without saying that such modifications arepreferred, which do not impair RNA stability, e.g. in comparison to thenucleic acid from which it is derived.

“Different Hendra virus”, “different Nipah virus”, “differentHenipavirus”: The terms “different Hendra virus”, “different Nipahvirus”, “different Henipavirus” in the context of the invention has tobe understood as the difference between at least two respective viruses,wherein the difference is manifested on the RNA genome of the respectivedifferent virus. In the broadest sense, “different Nipah virus” has tobe understood as genetically “different Nipah virus”. Similarly,“different Hendra virus” has to be understood as genetically “differentHendra virus”. Particularly, said (genetically) different virusesexpress at least one different protein or peptide, wherein the at leastone different protein or peptide preferably differs in at least oneamino acid.

“Same Henipavirus”, “same Nipah virus”, “same Hendra virus”: In thebroadest sense, “same Henipavirus”, “same Nipah virus”, or “same Hendravirus” has to be understood as genetically. Particularly, said(genetically) same virus expresses the same proteins or peptides (e.g.,at least one structural and/or non-structural protein), wherein allproteins or peptides have the same amino acid sequence.

DNA: DNA is the usual abbreviation for deoxy-ribonucleic acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually deoxy-adenosine-monophosphate,deoxy-thymidine-monophosphate, deoxy-guanosine-monophosphate anddeoxy-cytidine-monophosphate monomers which are—by themselves—composedof a sugar moiety (deoxyribose), a base moiety and a phosphate moiety,and polymerize by a characteristic backbone structure. The backbonestructure is, typically, formed by phosphodiester bonds between thesugar moiety of the nucleotide, i.e. deoxyribose, of a first and aphosphate moiety of a second, adjacent monomer. The specific order ofthe monomers, i.e. the order of the bases linked to thesugar/phosphate-backbone, is called the DNA sequence. DNA may be singlestranded or double stranded. In the double stranded form, thenucleotides of the first strand typically hybridize with the nucleotidesof the second strand, e.g. by NT-base-pairing and G/C-base-pairing.

Epitope: An “epitope” (also called “antigen determinant”) can bedistinguished in T cell epitopes and B cell epitopes. T cell epitopes orparts of the proteins in the context of the present invention maycomprise fragments preferably having a length of about 6 to about 20 oreven more amino acids, e.g. fragments as processed and presented by MHCclass I molecules, preferably having a length of about 8 to about 10amino acids, e.g. 8, 9, or 10, (or even 11, or 12 amino acids), orfragments as processed and presented by MHC class II molecules,preferably having a length of about 13 or more amino acids, e.g. 13, 14,15, 16, 17, 18, 19, 20 or even more amino acids, wherein these fragmentsmay be selected from any part of the amino acid sequence. Thesefragments are typically recognized by T cells in form of a complexconsisting of the peptide fragment and an MHC molecule, i.e. thefragments are typically not recognized in their native form. B cellepitopes are typically fragments located on the outer surface of(native) protein or peptide antigens as defined herein, preferablyhaving 5 to 15 amino acids, more preferably having 5 to 12 amino acids,even more preferably having 6 to 9 amino acids, which may be recognizedby antibodies, i.e. in their native form. Such epitopes of proteins orpeptides may furthermore be selected from any of the herein mentionedvariants of such proteins or peptides. In this context antigenicdeterminants can be conformational or discontinuous epitopes which arecomposed of segments of the proteins or peptides as defined herein thatare discontinuous in the amino acid sequence of the proteins or peptidesas defined herein but are brought together in the three-dimensionalstructure or continuous or linear epitopes which are composed of asingle polypeptide chain.

Fragment of a sequence: A fragment of a sequence may typically be ashorter portion of a full-length sequence of e.g. a nucleic acidsequence or an amino acid sequence. Accordingly, a fragment, typically,consists of a sequence that is identical to the corresponding stretchwithin the full-length sequence. A preferred fragment of a sequence inthe context of the present invention, consists of a continuous stretchof entities, such as nucleotides or amino acids corresponding to acontinuous stretch of entities in the molecule the fragment is derivedfrom, which represents at least 5%, 10%, 20%, preferably at least 30%,more preferably at least 40%, more preferably at least 50%, even morepreferably at least 60%, even more preferably at least 70%, and mostpreferably at least 80% of the total (i.e. full-length) molecule fromwhich the fragment is derived.

Fragments of proteins: “Fragments” of proteins or peptides in thecontext of the present invention may, typically, comprise a sequence ofa protein or peptide as defined herein, which is, with regard to itsamino acid sequence (or its encoded nucleic acid molecule), N-terminallyand/or C-terminally truncated compared to the amino acid sequence of theoriginal (native) protein (or its encoded nucleic acid molecule). Suchtruncation may thus occur either on the amino acid level orcorrespondingly on the nucleic acid level. A sequence identity withrespect to such a fragment as defined herein may therefore preferablyrefer to the entire protein or peptide as defined herein or to theentire (coding) nucleic acid molecule of such a protein or peptide. Inthe context of antigens such fragment may have a length of about 6 toabout 20 or even more amino acids, e.g. fragments as processed andpresented by MHC class I molecules, preferably having a length of about8 to about 10 amino acids, e.g. 8, 9, or 10, (or even 6, 7, 11, or 12amino acids), or fragments as processed and presented by MHC class IImolecules, preferably having a length of about 13 or more amino acids,e.g. 13, 14, 15, 16, 17, 18, 19, 20 or even more amino acids, whereinthese fragments may be selected from any part of the amino acidsequence. These fragments are typically recognized by T-cells in form ofa complex consisting of the peptide fragment and an MHC molecule, i.e.the fragments are typically not recognized in their native form.Fragments of proteins or peptides (e.g. in the context of antigens) maycomprise at least one epitope of those proteins or peptides. Furthermorealso domains of a protein, like the extracellular domain, theintracellular domain or the transmembrane domain and shortened ortruncated versions of a protein may be understood to comprise a fragmentof a protein.

G/C modified: The terms “G/C modified” or “G/C content modification” maytypically be a nucleic acid, preferably an artificial nucleic acidmolecule as defined herein, based on a modified wild type sequencecomprising a preferably increased number of guanosine and/or cytosinenucleotides as compared to the wild type sequence. Such an increasednumber may be generated by substitution of codons containing adenosineor thymidine nucleotides by codons containing guanosine or cytosinenucleotides. If the enriched G/C content occurs in a coding sequence ofDNA or RNA, it makes use of the degeneracy of the genetic code.Accordingly, the codon substitutions preferably do not alter the encodedamino acid residues, but exclusively increase the G/C content of thenucleic acid molecule.

Gene therapy: Gene therapy may typically be understood to mean atreatment of a patient's body or isolated elements of a patient's body,for example isolated tissues/cells, by nucleic acids encoding a peptideor protein. It typically may comprise at least one of the steps of a)administration of a nucleic acid, preferably an artificial nucleic acidmolecule as defined herein, directly to the patient—by whateveradministration route—or in vitro to isolated cells/tissues of thepatient, which results in transfection of the patient's cells either invivo/ex vivo or in vitro; b) transcription and/or translation of theintroduced nucleic acid molecule; and optionally c) re-administration ofisolated, transfected cells to the patient, if the nucleic acid has notbeen administered directly to the patient.

Genetic vaccination: Genetic vaccination may typically be understood tobe vaccination by administration of a nucleic acid molecule encoding anantigen or an immunogen or fragments thereof. The nucleic acid moleculemay be administered to a subject's body or to isolated cells of asubject. Upon transfection of certain cells of the body or upontransfection of the isolated cells, the antigen or immunogen may beexpressed by those cells and subsequently presented to the immunesystem, eliciting an adaptive, i.e. antigen-specific immune response.Accordingly, genetic vaccination typically comprises at least one of thesteps of a) administration of a nucleic acid, preferably an artificialnucleic acid molecule as defined herein, to a subject, preferably apatient, or to isolated cells of a subject, preferably a patient, whichusually results in transfection of the subject's cells either in vivo orin vitro; b) transcription and/or translation of the introduced nucleicacid molecule; and optionally c) re-administration of isolated,transfected cells to the subject, preferably the patient, if the nucleicacid has not been administered directly to the patient.

Genotype, genotype of a virus: The genetic constitution of an individualor a group or class of organisms having the same genetically consistentstructure. Genotyping means determining differences in the genetic of anindividual. In the context of the invention, Nipah virus genotype has tobe understood as a Nipah virus having the same genetically consistentstructure and Hendra virus genotype has to be understood as a Hendravirus having the same genetically consistent structure.

Heterologous sequence: Two sequences are typically understood to be“heterologous” if they are not derivable from the same gene or in thesame allele. I.e., although heterologous sequences may be derivable fromthe same organism, they naturally (in nature) do not occur in the samenucleic acid molecule, such as in the same mRNA.

Homolog of a nucleic acid sequence: The term “homolog” of a nucleic acidsequence refers to sequences of other species than the particularsequence. E.g., in the context of the invention, it is particularlypreferred that the nucleic acid sequence is derived from a Nipah virusand/or Hendra virus; therefore it is preferred that the homolog is ahomolog of a respective Nipah virus or respective Hendra virus nucleicacid sequence.

Humoral immunity/humoral immune response: Humoral immunity referstypically to B-cell mediated antibody production and optionally toaccessory processes accompanying antibody production. A humoral immuneresponse may be typically characterized, e.g., by Th2 activation andcytokine production, germinal center formation and isotype switching,affinity maturation and memory cell generation. Humoral immunity alsotypically may refer to the effector functions of antibodies, whichinclude pathogen and toxin neutralization, classical complementactivation, and opsonin promotion of phagocytosis and pathogenelimination.

“Hybridizing” or “Hybridizing with a complement sequence”: Nucleic acidmolecules which are advantageously for the process according to theinvention can be isolated based on their homology to the nucleic acidmolecules or a complement sequence of the nucleic acid moleculesdisclosed herein using the sequences or part thereof as hybridizationprobe and following standard hybridization techniques under stringenthybridization conditions. In this context, it is possible to use, forexample, isolated nucleic acid molecules of at least 15, 20, 25, 30, 35,40, 50, 60 or more nucleotides, preferably of at least 15, 20 or 25nucleotides in length which hybridize under stringent conditions withthe above-described nucleic acid molecules, in particular with thosewhich encompass a nucleotide sequence of the nucleic acid molecule usedin the invention or encoding a protein used in the invention or of thenucleic acid molecule of the invention. Nucleic acid molecules with 30,50, 100, 250 or more nucleotides may also be used. The term “homology”means that the respective nucleic acid molecules or encoded proteins arefunctionally and/or structurally equivalent. The nucleic acid moleculesthat are homologous to the nucleic acid molecules described above andthat are derivatives of said nucleic acid molecules are, for example,variations of said nucleic acid molecules which represent modificationshaving the same biological function, in particular encoding proteinswith the same or substantially the same biological function. They may benaturally occurring variations, such as sequences from other species,strains, or mutations. These mutations may occur naturally or may beobtained by mutagenesis techniques. The allelic variations may benaturally occurring allelic variants as well as synthetically producedor genetically engineered variants. Structurally equivalents can, forexample, be identified by testing the binding of said polypeptide toantibodies or computer based predictions. By “hybridizing” it is meantthat such nucleic acid molecules hybridize under conventionalhybridization conditions, preferably under stringent conditions such asdescribed by, e.g., Sambrook (Molecular Cloning; A Laboratory Manual,2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y. (1989)) or in Current Protocols in Molecular Biology, John Wiley &Sons, N.Y. (1989), 6.3.1-6.3.6. According to the invention, DNA as wellas RNA molecules of the nucleic acid of the invention can be used asprobes. Further, as template for the identification of functionalhomologues Northern blot assays as well as Southern blot assays can beperformed. The Northern blot assay advantageously provides furtherinformation about the expressed gene product: e.g. expression pattern,occurrence of processing steps, like splicing and capping, etc. TheSouthern blot assay provides additional information about thechromosomal localization and organization of the gene encoding thenucleic acid molecule of the invention. A preferred, no limiting exampleof stringent hybridization conditions are hybridizations in 6× sodiumchloride/sodium citrate (═SSC) at approximately 45° C., followed by oneor more wash steps in 0.2×SSC, 0.1% SDS at 50 to 65° C., for example at50° C., 55° C. or 60° C. The skilled worker knows that thesehybridization conditions differ as a function of the type of the nucleicacid and, for example when organic solvents are present, with regard tothe temperature and concentration of the buffer. The temperature under“standard hybridization conditions” differs for example as a function ofthe type of the nucleic acid between 42° C. and 58° C., preferablybetween 45° C. and 50° C. in an aqueous buffer with a concentration of0.1× 0.5×, 1×, 2×, 3×, 4× or 5×SSC (pH 7.2). If organic solvent(s)is/are present in the abovementioned buffer, for example 50% formamide,the temperature under standard conditions is approximately 40° C., 42°C. or 45° C. The hybridization conditions for DNA:DNA hybrids arepreferably for example 0.1×SSC and 20° C., 25° C., 30° C., 35° C., 40°C. or 45° C., preferably between 30° C. and 45° C. The hybridizationconditions for DNA:RNA hybrids are preferably for example 0.1×SSC and30° C., 35° C., 40° C., 45° C., 50° C. or 55° C., preferably between 45°C. and 55° C. The abovementioned hybridization temperatures aredetermined for example for a nucleic acid approximately 100 bp (=basepairs) in length and a G+C content of 50% in the absence of formamide.The skilled worker knows to determine the hybridization conditionsrequired with the aid of textbooks, for example the ones mentionedabove, or from the following textbooks: Sambrook et al., “MolecularCloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.)1985, “Nucleic Acids Hybridization: A Practical Approach”, IRL Press atOxford University Press, Oxford; Brown (Ed.) 1991, “Essential MolecularBiology: A Practical Approach”, IRL Press at Oxford University Press,Oxford. A further example of one such stringent hybridization conditionis hybridization at 4×SSC at 65° C., followed by a washing in 0.1×SSC at65° C. for one hour. Alternatively, an exemplary stringent hybridizationcondition is in 50% formamide, 4×SSC at 42° C. Further, the conditionsduring the wash step can be selected from the range of conditionsdelimited by low-stringency conditions (approximately 2×SSC at 50° C.)and high-stringency conditions (approximately 0.2×SSC at 50° C.,preferably at 65° C.) (20×SSC: 0.3M sodium citrate, 3M NaCl, pH 7.0). Inaddition, the temperature during the wash step can be raised fromlow-stringency conditions at room temperature, approximately 22° C., tohigher-stringency conditions at approximately 65° C. Both of theparameters salt concentration and temperature can be variedsimultaneously, or else one of the two parameters can be kept constantwhile only the other is varied. Denaturants, for example formamide orSDS, may also be employed during the hybridization. In the presence of50% formamide, hybridization is preferably effected at 42° C. Relevantfactors like i) length of treatment, ii) salt conditions, iii) detergentconditions, iv) competitor DNAs, v) temperature and vi) probe selectioncan be combined case by case so that not all possibilities can bementioned herein.

Some examples of conditions for DNA hybridization (Southern blot assays)and wash step are shown herein below:

(1) Hybridization conditions can be selected, for example, from thefollowing conditions:

a) 4×SSC at 65° C.,

b) 6×SSC at 45° C.,

c) 6×SSC, 100 mg/ml denatured fragmented fish sperm DNA at 68° C.,

d) 6×SSC, 0.5% SDS, 100 mg/ml denatured salmon sperm DNA at 68° C.,

e) 6×SSC, 0.5% SDS, 100 mg/ml denatured fragmented salmon sperm DNA, 50%formamide at 42° C.,

f) 50% formamide, 4×SSC at 42° C.,

g) 50% (vol/vol) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1%polyvinylpyrrolidone, 50 mM sodium phosphate buffer pH 6.5, 750 mM NaCl,75 mM sodium citrate at 42° C.,

h) 2× or 4×SSC at 50° C. (low-stringency condition), or

i) 30 to 40% formamide, 2× or 4×SSC at 42° C. (low-stringencycondition).

(2) Wash steps can be selected, for example, from the followingconditions:

a) 0.015 M NaCl/0.0015 M sodium citrate/0.1% SDS at 50° C.

b) 0.1×SSC at 65° C.

c) 0.1×SSC, 0.5% SDS at 68° C.

d) 0.1×SSC, 0.5% SDS, 50% formamide at 42° C.

e) 0.2×SSC, 0.1% SDS at 42° C.

f) 2×SSC at 65° C. (low-stringency condition).

Further, some applications have to be performed at low stringencyhybridization conditions, without any consequences for the specificityof the hybridization. For example, a Southern blot analysis of total DNAcould be probed with a nucleic acid molecule of the present inventionand washed at low stringency (55° C. in 2×SSPE, 0.1% SDS). A furtherexample of such low-stringent hybridization conditions is 4×SSC at 50°C. or hybridization with 30 to 40% formamide at 42° C. Such moleculescomprise those which are fragments, analogues or derivatives of thepolypeptide of the invention or used in the methods of the invention anddiffer, for example, by way of amino acid and/or nucleotide deletion(s),insertion(s), substitution (s), addition(s) and/or recombination (s) orany other modification(s) known in the art either alone or incombination from the above-described amino acid sequences or theirunderlying nucleotide sequence(s). However, it is preferred to use highstringency hybridization conditions. Hybridization should advantageouslybe carried out with fragments of at least 5, 10, 15, 20, 25, 30, 35 or40 bp, advantageously at least 50, 60, 70 or 80 bp, preferably at least90, 100 or 110 bp. Most preferably are fragments of at least 15, 20, 25or 30 bp. Preferably are also hybridizations with at least 100 bp or200, very especially preferably at least 400 bp in length. In anespecially preferred embodiment, the hybridization should be carried outwith the entire nucleic acid sequence with conditions described above.The term “hybridizes under stringent conditions” is defined above. Inone embodiment, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences at least 30%, 40%, 50% or 65% identical toeach other typically remain hybridized to each other. Preferably, theconditions are such that sequences at least about 70%, more preferablyat least about 75% or 80%, and even more preferably at least about 85%,90% or 95% or more identical to each other typically remain hybridizedto each other. To determine the percentage homology (=identity, hereinused interchangeably) of two amino acid sequences or of two nucleic acidmolecules, the sequences are written one underneath the other for anoptimal comparison (for example gaps may be inserted into the sequenceof a protein or of a nucleic acid in order to generate an optimalalignment with the other protein or the other nucleic acid). The aminoacid residues or nucleic acid molecules at the corresponding amino acidpositions or nucleotide positions are then compared. If a position inone sequence is occupied by the same amino acid residue or the samenucleic acid molecule as the corresponding position in the othersequence, the molecules are homologous at this position (i.e. amino acidor nucleic acid “homology” as used in the present context corresponds toamino acid or nucleic acid “identity”. The percentage homology betweenthe two sequences is a function of the number of identical positionsshared by the sequences (i.e. % homology=number of identicalpositions/total number of positions×100). The terms “homology” and“identity” are thus to be considered as synonyms. For the determinationof the percentage homology (=identity) of two or more amino acids or oftwo or more nucleotide sequences several computer software programs havebeen developed. The homology of two or more sequences can be calculatedwith for example the software fasta, which presently has been used inthe version fasta 3 (W. R. Pearson and D. J. Lipman (1988), ImprovedTools for Biological Sequence Comparison. PNAS 85:2444-2448; W. R.Pearson (1990) Rapid and Sensitive Sequence Comparison with FASTP andFASTA, Methods in Enzymology 183:63-98; W. R. Pearson and D. J. Lipman(1988) Improved Tools for Biological Sequence Comparison. PNAS85:2444-2448; W. R. Pearson (1990); Rapid and Sensitive SequenceComparison with FASTP and FASTAMethods in Enzymology 183:63-98). Anotheruseful program for the calculation of homologies of different sequencesis the standard blast program, which is included in the Biomax pedantsoftware (Biomax, Munich, Federal Republic of Germany). This leadsunfortunately sometimes to suboptimal results since blast does notalways include complete sequences of the subject and the query.Nevertheless as this program is very efficient it can be used for thecomparison of a huge number of sequences. The following settings aretypically used for such a comparisons of sequences: −p Program Name[String]; −d Database [String]; default=nr; −i Query File [File In];default=stdin; −e Expectation value (E) [Real]; default=10.0; −malignment view options: 0=pairwise; 1=query-anchored showing identities;2=query-anchored no identities; 3=flat query-anchored, show identities;4=flat query-anchored, no identities; 5=query-anchored no identities andblunt ends; 6=flat query-anchored, no identities and blunt ends; 7=XMLBlast output; 8=tabular; 9 tabular with comment lines [Integer];default=0; −o BLAST report Output File [File Out] Optional;default=stdout; −F Filter query sequence (DUST with blastn, SEG withothers) [String]; default=T; −G Cost to open a gap (zero invokes defaultbehavior) [Integer]; default=0; −E Cost to extend a gap (zero invokesdefault behavior) [Integer]; default=0; −X X dropoff value for gappedalignment (in bits) (zero invokes default behavior); blastn 30,megablast 20, tblastx 0, all others 15 [Integer]; default=0; −l ShowGl's in deflines [T/F]; default=F; −q Penalty for a nucleotide mismatch(blastn only) [Integer]; default=−3; −r Reward for a nucleotide match(blastn only) [Integer]; default=1; −v Number of database sequences toshow one-line descriptions for (V) [Integer]; default=500; −b Number ofdatabase sequence to show alignments for (B) [Integer]; default=250; −fThreshold for extending hits, default if zero; blastp 11, blastn 0,blastx 12, tblastn 13; tblastx 13, megablast 0 [Integer]; default=0; −gPerfom gapped alignment (not available with tblastx) [T/F]; default=T;−Q Query Genetic code to use [Integer]; default=1; −D DB Genetic code(for tblast[nx] only) [Integer]; default=1; −a Number of processors touse [Integer]; default=1; −O SeqAlign file [File Out] Optional; −JBelieve the query define [T/F]; default=F; −M Matrix [String];default=BLOSUM62; −W Word size, default if zero (blastn 11, megablast28, all others 3) [Integer]; default=0; −z Effective length of thedatabase (use zero for the real size) [Real]; default=0; −K Number ofbest hits from a region to keep (off by default, if used a value of 100is recommended) [Integer]; default=0; −P 0 for multiple hit, 1 forsingle hit [Integer]; default=0; −Y Effective length of the search space(use zero for the real size) [Real]; default=0; −S Query strands tosearch against database (for blast[nx], and tblastx); 3 is both, 1 istop, 2 is bottom [Integer]; default=3; −T Produce HTML output [T/F];default=F; −l Restrict search of database to list of GI's [String]Optional; −U Use lower case filtering of FASTA sequence [T/F] Optional;default=F; −y X dropoff value for ungapped extensions in bits (0.0invokes default behavior); blastn 20, megablast 10, all others 7 [Real];default=0.0; −Z X dropoff value for final gapped alignment in bits (0.0invokes default behavior); blastn/megablast 50, tblastx 0, all others 25[Integer]; default=0; −R PSI-TBLASTN checkpoint file [File In] Optional;−n MegaBlast search [T/F]; default=F; −L Location on query sequence[String] Optional; −A Multiple Hits window size, default if zero(blastn/megablast 0, all others 40 [Integer]; default=0; −w Frame shiftpenalty (OOF algorithm for blastx) [Integer]; default=0; −t Length ofthe largest intron allowed in tblastn for linking HSPs (0 disableslinking) [Integer]; default=0.

Results of high quality are reached by using the algorithm of Needlemanand Wunsch or Smith and Waterman. Therefore programs based on saidalgorithms are preferred. Advantageously the comparisons of sequencescan be done with the program PileUp (J. Mol. Evolution., 25, 351 (1987),Higgins et al., CABIOS 5, 151 (1989)) or preferably with the programs“Gap” and “Needle”, which are both based on the algorithms of Needlemanand Wunsch (J. Mol, Biol. 48; 443 (1970)), and “BestFit”, which is basedon the algorithm of Smith and Waterman (Adv. Appl. Math. 2; 482 (1981)).“Gap” and “BestFit” are part of the GCG software-package (GeneticsComputer Group, 575 Science Drive, Madison, Wis., USA 53711 (1991);Altschul et al., (Nucleic Acids Res. 25, 3389 (1997)), “Needle” is partof the The European Molecular Biology Open Software Suite (EMBOSS)(Trends in Genetics 16 (6), 276 (2000)). Therefore preferably thecalculations to determine the percentages of sequence homology are donewith the programs “Gap” or “Needle” over the whole range of thesequences. The following standard adjustments for the comparison ofnucleic acid sequences were used for “Needle”: matrix: EDNAFULL,Gap_penalty: 10.0, Extend_penalty: 0.5. The following standardadjustments for the comparison of nucleic acid sequences were used for“Gap”: gap weight: 50, length weight: 3, average match: 10.000, averagemismatch: 0.000.

Identity of a sequence: In order to determine the percentage to whichtwo sequences are identical, e.g. nucleic acid sequences or amino acidsequences as defined herein, preferably the amino acid sequences encodedby a nucleic acid sequence of the polymeric carrier as defined herein orthe amino acid sequences themselves, the sequences can be aligned inorder to be subsequently compared to one another. Therefore, e.g. aposition of a first sequence may be compared with the correspondingposition of the second sequence. If a position in the first sequence isoccupied by the same component (residue) as is the case at a position inthe second sequence, the two sequences are identical at this position.If this is not the case, the sequences differ at this position. Ifinsertions occur in the second sequence in comparison to the firstsequence, gaps can be inserted into the first sequence to allow afurther alignment. If deletions occur in the second sequence incomparison to the first sequence, gaps can be inserted into the secondsequence to allow a further alignment. The percentage to which twosequences are identical is then a function of the number of identicalpositions divided by the total number of positions including thosepositions which are only occupied in one sequence. The percentage towhich two sequences are identical can be determined using a mathematicalalgorithm. A preferred, but not limiting, example of a mathematicalalgorithm which can be used is the algorithm of Karlin et al. (1993),PNAS USA, 90:5873-5877 or Altschul et al. (1997), Nucleic Acids Res.,25:3389-3402. Such an algorithm is integrated in the BLAST program.Sequences which are identical to the sequences of the present inventionto a certain extent can be identified by this program.

Immunogen: In the context of the present invention an immunogen may betypically understood to be a compound that is able to stimulate animmune response. Preferably, an immunogen is a peptide, polypeptide, orprotein. In a particularly preferred embodiment, an immunogen in thesense of the present invention is the product of translation of aprovided nucleic acid molecule, preferably an artificial nucleic acidmolecule as defined herein. Typically, an immunogen elicits at least anadaptive immune response.

Immunostimulatory composition: In the context of the invention, animmunostimulatory composition may be typically understood to be acomposition containing at least one component which is able to induce animmune response or from which a component which is able to induce animmune response is derivable. Such immune response may be preferably aninnate immune response or a combination of an adaptive and an innateimmune response. Preferably, an immunostimulatory composition in thecontext of the invention contains at least one artificial nucleic acidmolecule, more preferably an RNA, for example an mRNA molecule. Theimmunostimulatory component, such as the mRNA may be complexed with asuitable carrier. Thus, the immunostimulatory composition may comprisean mRNA/carrier-complex. Furthermore, the immunostimulatory compositionmay comprise an adjuvant and/or a suitable vehicle for theimmunostimulatory component, such as the mRNA.

Immunostimulatory RNA: An immunostimulatory RNA (isRNA) in the contextof the invention may typically be an RNA that is able to induce aninnate immune response itself. It usually does not have an open readingframe and thus does not provide a peptide-antigen or immunogen butelicits an innate immune response e.g. by binding to a specific kind ofToll-like-receptor (TLR) or other suitable receptors. However, of coursealso mRNAs having an open reading frame and coding for a peptide/protein(e.g. an antigenic function) may induce an innate immune response and,thus, may be immunostimulatory RNAs.

Immune response: An immune response may typically be a specific reactionof the adaptive immune system to a particular antigen (so calledspecific or adaptive immune response) or an unspecific reaction of theinnate immune system (so called unspecific or innate immune response),or a combination thereof.

Immune system: The immune system may protect organisms from infection.If a pathogen succeeds in passing a physical barrier of an organism andenters this organism, the innate immune system provides an immediate,but non-specific response. If pathogens evade this innate response,vertebrates possess a second layer of protection, the adaptive immunesystem. Here, the immune system adapts its response during an infectionto improve its recognition of the pathogen. This improved response isthen retained after the pathogen has been eliminated, in the form of animmunological memory, and allows the adaptive immune system to mountfaster and stronger attacks each time this pathogen is encountered.According to this, the immune system comprises the innate and theadaptive immune system. Each of these two parts typically contains socalled humoral and cellular components.

Innate immune system: The innate immune system, also known asnon-specific (or unspecific) immune system, typically comprises thecells and mechanisms that defend the host from infection by otherorganisms in a non-specific manner. This means that the cells of theinnate system may recognize and respond to pathogens in a generic way,but unlike the adaptive immune system, it does not confer long-lastingor protective immunity to the host. The innate immune system may be,e.g., activated by ligands of Toll-like receptors (TLRs) or otherauxiliary substances such as lipopolysaccharides, TNF-alpha, CD40ligand, or cytokines, monokines, lymphokines, interleukins orchemokines, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,M-CSF, LT-beta, TNF-alpha, growth factors, and hGH, a ligand of humanToll-like receptor TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, a ligand of murine Toll-like receptor TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13, a ligand ofa NOD-like receptor, a ligand of a RIG-I like receptor, animmunostimulatory nucleic acid, an immunostimulatory RNA (isRNA), aCpG-DNA, an antibacterial agent, or an anti-viral agent. Thepharmaceutical composition according to the present invention maycomprise one or more such substances. Typically, a response of theinnate immune system includes recruiting immune cells to sites ofinfection, through the production of chemical factors, includingspecialized chemical mediators, called cytokines; activation of thecomplement cascade; identification and removal of foreign substancespresent in organs, tissues, the blood and lymph, by specialized whiteblood cells; activation of the adaptive immune system; and/or acting asa physical and chemical barrier to infectious agents.

Jet injection: The term “jet injection”, as used herein, refers to aneedle-free injection method, wherein a fluid containing at least oneinventive nucleic acid sequence (e.g., RNA, DNA, mRNA) and, optionally,further suitable excipients is forced through an orifice, thusgenerating an ultra-fine liquid stream of high pressure that is capableof penetrating mammalian skin and, depending on the injection settings,subcutaneous tissue or muscle tissue. In principle, the liquid streamforms a hole in the skin, through which the liquid stream is pushed intothe target tissue. Preferably, jet injection is used for intradermal,subcutaneous or intramuscular injection of the mRNA sequence accordingto the invention. In a preferred embodiment, jet injection is used forintramuscular injection of the mRNA sequence according to the invention.In a further preferred embodiment, jet injection is used for intradermalinjection of the mRNA sequence according to the invention.

Monocistronic nucleic acid: A monocistronic nucleic acid may typicallybe a DNA or RNA, particularly an mRNA that comprises only one codingsequences. A coding sequence in this context is a sequence of severalnucleotide triplets (codons) that can be translated into a peptide orprotein.

Monovalent/monovalent vaccine: A monovalent vaccine, also calledunivalent vaccine, is designed against a single antigen for a singleorganism. The term “monovalent vaccine” includes the immunizationagainst a single valence. In the context of the invention, a monovalentNipah virus vaccine would comprise a vaccine comprising an artificialnucleic acid encoding one single antigenic peptide or protein derivedfrom one specific Nipah virus strain and a monovalent Hendra virusvaccine would comprise a vaccine comprising an artificial nucleic acidencoding one single antigenic peptide or protein derived from onespecific Hendra virus strain

Nucleic acid molecule: A nucleic acid molecule is a molecule comprising,preferably consisting of nucleic acid components. The term nucleic acidmolecule preferably refers to DNA or RNA molecules. It is preferablyused synonymous with the term “polynucleotide”. Preferably, a nucleicacid molecule is a polymer comprising or consisting of nucleotidemonomers, which are covalently linked to each other byphosphodiester-bonds of a sugar/phosphate-backbone. The term “nucleicacid molecule” also encompasses modified nucleic acid molecules, such asbase-modified, sugar-modified or backbone-modified etc. DNA or RNAmolecules.

Nucleic acid sequence/amino acid: The sequence of a nucleic acidmolecule is typically understood to be the particular and individualorder, i.e. the succession of its nucleotides. The sequence of a proteinor peptide is typically understood to be the order, i.e. the successionof its amino acids.

Orthologues and paralogues: Orthologues and paralogues (of a sequence)encompass evolutionary concepts used to describe the ancestralrelationships of genes and their corresponding gene products (proteins).Paralogues are genes (or proteins) within the same species that haveoriginated through duplication of an ancestral gene; orthologues aregenes (or proteins) from different organisms that have originatedthrough speciation, and are also derived from a common ancestral gene.In the context of the invention, an orthologue and/or a paralogue of aNipah virus nucleic acid sequence of the invention refers to a sequencehaving in increasing order of preference at least 50%, 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% or more sequence identity to the sequence asrepresented by SEQ ID NOs: 27-33, 38-44, 53-59, 64-70, 79-85, 90-96,105-111, 116-122, 131-137, 142-148, 157-163, 168-174, 183-189, 194-200,209-215, 220-226, 599-605, 625-631, 651-657, 677-683, 703-709, 729-735,755-761, 781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943, 963-969,989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928, 948-954,974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099, 1119-1125,1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255, 1078-1084,1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214, 1234-1240,1260-1266, 1275-1281, 1286-1292, 1301-1307, 1312-1318, 1327-1333,1338-1344, 1353-1359, 1364-1370, 1379-1385, 1390-1396, 1405-1411,1416-1422, 1431-1437, 1457-1463, 1483-1489, 1442-1448, 1468-1474,1494-1500, 1516-1539, 1540-1548. In the context of the invention, anorthologue and/or a paralogue of a Nipah virus amino acid sequence ofthe invention refers to a sequence having in increasing order ofpreference at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or moresequence identity to the sequence as represented by SEQ ID NOs: 1-7,12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047, 1052-1058,1513-1515. In the context of the invention, an orthologue and/or aparalogue of a Hendra virus nucleic acid sequence of the inventionrefers to a sequence having in increasing order of preference at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity tothe sequence as represented by SEQ ID NOs: 34-37, 45-52, 60-63, 71-78,86-89, 97-104, 112-115, 123-130, 138-141, 149-156, 164-167, 175-182,190-193, 201-208, 216-219, 227-234, 606-609, 632-635, 658-661, 684-687,710-713, 736-739, 762-765, 788-791, 617-624, 643-650, 669-676, 695-702,721-728, 747-754, 773-780, 799-806, 840-843, 866-869, 892-895, 918-921,944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884, 903-910,929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077, 1100-1103,1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259,1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196, 1215-1222,1241-1248, 1267-1274, 1282-1285, 1293-1300, 1308-1311, 1319-1326,1334-1337, 1345-1352, 1360-1363, 1371-1378, 1386-1389, 1397-1404,1412-1415, 1423-1430, 1438-1441, 1464-1467, 1490-1493, 1449-1456,1475-1482, 1501-1508. In the context of the invention, an orthologueand/or a paralogue of a Hendra virus amino acid sequence of theinvention refers to a sequence having in increasing order of preferenceat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequenceidentity to the sequence as represented by SEQ ID NOs: 8-11, 19-26,580-583, 591-598, 814-817, 825-832, 1048-1051, 1059-1066.

Peptide: A peptide or polypeptide is typically a polymer of amino acidmonomers, linked by peptide bonds. It typically contains less than 50monomer units. Nevertheless, the term peptide is not a disclaimer formolecules having more than 50 monomer units. Long peptides are alsocalled polypeptides, typically having between 50 and 600 monomericunits. The term “polypeptide” as used herein, however, is typically notlimited by the length of the molecule it refers to. In the context ofthe present invention, the term “polypeptide” may also be used withrespect to peptides comprising less than 50 (e.g., 10) amino acids orpeptides comprising even more than 600 amino acids.

Pharmaceutically effective amount: A pharmaceutically effective amountin the context of the invention is typically understood to be an amountthat is sufficient to induce a pharmaceutical effect, such as an immuneresponse, altering a pathological level of an expressed peptide orprotein, or substituting a lacking gene product, e.g., in case of apathological situation.

Protein: A protein typically comprises one or more peptides orpolypeptides. A protein is typically folded into 3-dimensional form,which may be required for the protein to exert its biological function.

Poly(A) sequence: A poly(A) sequence, also called poly(A) tail or3′-poly(A) tail, is typically understood to be a sequence of adenosinenucleotides, e.g., of up to about 400 adenosine nucleotides, e.g. fromabout 20 to about 400, preferably from about 50 to about 400, morepreferably from about 50 to about 300, even more preferably from about50 to about 250, most preferably from about 60 to about 250 adenosinenucleotides. A poly(A) sequence is typically located at the 3′-end of anmRNA. In the context of the present invention, a poly(A) sequence may belocated within an mRNA or any other nucleic acid molecule, such as,e.g., in a vector, for example, in a vector serving as template for thegeneration of an RNA, preferably an mRNA, e.g., by transcription of thevector. Moreover, poly(A) sequences, or poly(A) tails may be generatedin vitro by enzymatic polyadenylation of the RNA, e.g. usingPoly(A)polymerases (PAP) derived from Escherichia coli or yeast. Inaddition, polyadenylation of RNA can be achieved by using immobilizedPAP enzymes e.g. in a polyadenylation reactor (WO/2016/174271).

Poly(C) sequence: A poly(C) sequence is typically a long sequence ofcytosine nucleotides, typically about 10 to about 200 cytosinenucleotides, preferably about 10 to about 100 cytosine nucleotides, morepreferably about 10 to about 70 cytosine nucleotides or even more,preferably about 20 to about 50, or even about 20 to about 30 cytosinenucleotides. A poly(C) sequence may preferably be located 3′ of thecoding sequence comprised by a nucleic acid.

Polyadenylation: Polyadenylation is typically understood to be theaddition of a poly(A) sequence to a nucleic acid molecule, such as anRNA molecule, e.g. to a premature mRNA. Polyadenylation may be inducedby a so called polyadenylation signal. This signal is preferably locatedwithin a stretch of nucleotides at the 3′-end of a nucleic acidmolecule, such as an RNA molecule, to be polyadenylated. Apolyadenylation signal typically comprises a hexamer consisting ofadenine and uracil/thymine nucleotides, preferably the hexamer sequenceAAUAAA. Other sequences, preferably hexamer sequences, are alsoconceivable. Polyadenylation typically occurs during processing of apre-mRNA (also called premature-mRNA). Typically, RNA maturation (frompre-mRNA to mature mRNA) comprises the step of polyadenylation.

Polyvalent/polyvalent vaccine: A polyvalent vaccine, called alsomultivalent vaccine, containing antigens from more than one strain of avirus, or different antigens of the same virus, or any combinationthereof. The term “polyvalent vaccine” describes that this vaccine hasmore than one valence. In the context of the invention, a polyvalentNipah virus vaccine would comprise a vaccine comprising an artificialnucleic acid encoding antigenic peptides or proteins derived fromseveral different Nipah virus strains or comprising artificial nucleicacid encoding different antigens from the same Nipah virus strain, or acombination thereof. In preferred embodiment, a polyvalent Nipah virusvaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more differentartificial nucleic acids each encoding at least one different antigenicpeptide or protein. A polyvalent Hendra virus vaccine would comprise avaccine comprising an artificial nucleic acid encoding antigenicpeptides or proteins derived from several different Hendra virus strainsor comprising artificial nucleic acid encoding different antigens fromthe same Hendra virus strain, or a combination thereof. In preferredembodiment, a polyvalent Hendra virus vaccine comprises 2, 3, 4, 5, 6,7, 8, 9, 10 or even more different artificial nucleic acids eachencoding at least one different antigenic peptide or protein. Apolyvalent Henipavirus vaccine would comprise a vaccine comprising anartificial nucleic acid encoding antigenic peptides or proteins derivedfrom several different Henipavirus strains or comprising artificialnucleic acid encoding different antigens from the same Henipavirusstrain, or a combination thereof. In preferred embodiment, a polyvalentHenipavirus vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or even moredifferent artificial nucleic acids each encoding at least one differentantigenic peptide or protein (e.g., at least one derived from Nipahvirus and at least one derived from Hendra virus).

Restriction site: A restriction site, also termed restriction enzymerecognition site, is a nucleotide sequence recognized by a restrictionenzyme. A restriction site is typically a short, preferably palindromicnucleotide sequence, e.g. a sequence comprising 4 to 8 nucleotides. Arestriction site is preferably specifically recognized by a restrictionenzyme. The restriction enzyme typically cleaves a nucleotide sequencecomprising a restriction site at this site. In a double-strandednucleotide sequence, such as a double-stranded DNA sequence, therestriction enzyme typically cuts both strands of the nucleotidesequence.

RNA, mRNA: RNA is the usual abbreviation for ribonucleic-acid. It is anucleic acid molecule, i.e. a polymer consisting of nucleotides. Thesenucleotides are usually adenosine-monophosphate, uridine-monophosphate,guanosine-monophosphate and cytidine-monophosphate monomers which areconnected to each other along a so-called backbone. The backbone isformed by phosphodiester bonds between the sugar, i.e. ribose, of afirst and a phosphate moiety of a second, adjacent monomer. The specificsuccession of the monomers is called the RNA-sequence. Usually RNA maybe obtainable by transcription of a DNA-sequence, e.g., inside a cell.In eukaryotic cells, transcription is typically performed inside thenucleus or the mitochondria. Typically, transcription of DNA usuallyresults in the so-called premature RNA which has to be processed intoso-called messenger-RNA, usually abbreviated as mRNA. Processing of thepremature RNA, e.g. in eukaryotic organisms, comprises a variety ofdifferent posttranscriptional-modifications such as splicing,5′-capping, polyadenylation, export from the nucleus or the mitochondriaand the like. The sum of these processes is also called maturation ofRNA. The mature messenger RNA usually provides the nucleotide sequencethat may be translated into an amino-acid sequence of a particularpeptide or protein. Typically, a mature mRNA comprises a 5′-cap, a5′-UTR, a coding sequence, a 3′-UTR and a poly(A)sequence. Aside frommessenger RNA, several non-coding types of RNA exist which may beinvolved in regulation of transcription and/or translation.

RNA in vitro transcription: The terms “RNA in vitro transcription” or“in vitro transcription” relate to a process wherein RNA is synthesizedin a cell-free system (in vitro). DNA, particularly plasmid DNA, is usedas template for the generation of RNA transcripts. RNA may be obtainedby DNA-dependent in vitro transcription of an appropriate DNA template,which according to the present invention is preferably a linearizedplasmid DNA template. The promoter for controlling in vitrotranscription can be any promoter for any DNA-dependent RNA polymerase.Particular examples of DNA-dependent RNA polymerases are the T7, T3, andSP6 RNA polymerases. A DNA template for in vitro RNA transcription maybe obtained by cloning of a nucleic acid, in particular cDNAcorresponding to the respective RNA to be in vitro transcribed, andintroducing it into an appropriate vector for in vitro transcription,for example into plasmid DNA. In a preferred embodiment of the presentinvention the DNA template is linearized with a suitable restrictionenzyme, before it is transcribed in vitro. The cDNA may be obtained byreverse transcription of mRNA or chemical synthesis. Moreover, the DNAtemplate for in vitro RNA synthesis may also be obtained by genesynthesis. Methods for in vitro transcription are known in the art (see,e.g., Geall et al. (2013) Semin. Immunol. 25(2): 152-159; Brunelle etal. (2013) Methods Enzymol. 530:101-14). Reagents used in said methodtypically include:

1) a linearized DNA template with a promoter sequence that has a highbinding affinity for its respective RNA polymerase such asbacteriophage-encoded RNA polymerases;

2) ribonucleoside triphosphates (NTPs) for the four bases (adenine,cytosine, guanine and uracil); 3) optionally, a cap analogue as definedabove (e.g. m7G(5′)ppp(5′)G (m7G));

4) a DNA-dependent RNA polymerase capable of binding to the promotersequence within the linearized DNA template (e.g. T7, T3 or SP6 RNApolymerase);

5) optionally, a ribonuclease (RNase) inhibitor to inactivate anycontaminating RNase;

6) optionally, a pyrophosphatase to degrade pyrophosphate, which mayinhibit transcription;

7) MgCl₂, which supplies Mg²⁺ ions as a co-factor for the polymerase;

8) a buffer to maintain a suitable pH value, which can also containantioxidants (e.g. DTT), and/or polyamines such as spermidine at optimalconcentrations.

Sequence identity: Two or more sequences are identical if they exhibitthe same length and order of nucleotides or amino acids. The percentageof identity typically describes the extent, to which two sequences areidentical, i.e. it typically describes the percentage of nucleotidesthat correspond in their sequence position with identical nucleotides ofa reference sequence. In order to determine the degree of identity, thesequences to be compared are considered to exhibit the same length, i.e.the length of the longest sequence of the sequences to be compared. Thismeans that a first sequence consisting of 8 nucleotides is 80% identicalto a second sequence consisting of 10 nucleotides comprising the firstsequence. Hence, in the context of the present invention, identity ofsequences preferably relates to the percentage of nucleotides of asequence which have the same position in two or more sequences havingthe same length. Therefore, e.g. a position of a first sequence may becompared with the corresponding position of the second sequence. If aposition in the first sequence is occupied by the same component(residue) as is the case at a position in the second sequence, the twosequences are identical at this position. If this is not the case, thesequences differ at this position. If insertions occur in the secondsequence in comparison to the first sequence, gaps can be inserted intothe first sequence to allow a further alignment. If deletions occur inthe second sequence in comparison to the first sequence, gaps can beinserted into the second sequence to allow a further alignment. Thepercentage to which two sequences are identical is then a function ofthe number of identical positions divided by the total number ofpositions including those positions which are only occupied in onesequence. The percentage to which two sequences are identical can bedetermined using a mathematical algorithm. A preferred, but notlimiting, example of a mathematical algorithm which can be used is thealgorithm of Karlin et al. (1993), PNAS USA, 90:5873-5877 or Altschul etal. (1997), Nucleic Acids Res., 25:3389-3402. Such an algorithm isintegrated in the BLAST program. Sequences which are identical to thesequences of the present invention to a certain extent can be identifiedby this program.

Serotype, serotype of a virus: A serotype or a serotype of a virus is agroup of viruses classified together based on their antigens on thesurface of the virus, allowing the epidemiologic classification oforganisms to the sub-species level.

Strain, strain of a virus: A strain or a strain of a virus is a group ofviruses that are genetically distinct from other groups of the samespecies. The strain that is defined by a genetic variant is also definedas a “subtype”.

Stabilized nucleic acid molecule: A stabilized nucleic acid molecule isa nucleic acid molecule, preferably a DNA or RNA molecule that ismodified such, that it is more stable to disintegration or degradation,e.g., by environmental factors or enzymatic digest, such as by an exo-or endonuclease degradation, than the nucleic acid molecule without themodification. Preferably, a stabilized nucleic acid molecule in thecontext of the present invention is stabilized in a cell, such as aprokaryotic or eukaryotic cell, preferably in a mammalian cell, such asa human cell. The stabilization effect may also be exerted outside ofcells, e.g. in a buffer solution etc., for example, in a manufacturingprocess for a pharmaceutical composition comprising the stabilizednucleic acid molecule.

Transfection: The term “transfection” refers to the introduction ofnucleic acid molecules, such as DNA or RNA (e.g. mRNA) molecules, intocells, preferably into eukaryotic cells. In the context of the presentinvention, the term “transfection” encompasses any method known to theskilled person for introducing nucleic acid molecules into cells,preferably into eukaryotic cells, such as into mammalian cells. Suchmethods encompass, for example, electroporation, lipofection, e.g. basedon cationic lipids and/or liposomes, calcium phosphate precipitation,nanoparticle based transfection, virus based transfection, ortransfection based on cationic polymers, such as DEAE-dextran orpolyethylenimine etc. Preferably, the introduction is non-viral.

Vaccine: A vaccine is typically understood to be a prophylactic ortherapeutic material providing at least one antigen, preferably animmunogen. The antigen or immunogen may be derived from any materialthat is suitable for vaccination. For example, the antigen or immunogenmay be derived from a pathogen, such as from bacteria or virus particlesetc., or from a tumor or cancerous tissue. The antigen or immunogenstimulates the adaptive immune system of a mammalian subject to providean adaptive immune response. In the context of the present invention,the antigen is preferably provided via an artificial nucleic acid.

Variant of a nucleic acid sequence: A variant of a nucleic acid sequencerefers to a variant of nucleic acid sequences which forms the basis of anucleic acid sequence. For example, a variant nucleic acid sequence mayexhibit one or more nucleotide deletions, insertions, additions and/orsubstitutions compared to the nucleic acid sequence from which thevariant is derived. Preferably, a variant of a nucleic acid sequence isat least 40%, preferably at least 50%, more preferably at least 60%,more preferably at least 70%, even more preferably at least 80%, evenmore preferably at least 90%, most preferably at least 95% identical tothe nucleic acid sequence the variant is derived from. Preferably, thevariant is a functional variant. A “variant” of a nucleic acid sequencemay have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotideidentity over a stretch of 10, 20, 30, 50, 75 or 100 nucleotide of suchnucleic acid sequence.

Variants of proteins: “Variants” of proteins or peptides as defined inthe context of the present invention may be generated, having an aminoacid sequence which differs from the original sequence in one or moremutation(s), such as one or more substituted, inserted and/or deletedamino acid(s). Preferably, these fragments and/or variants have the samebiological function or specific activity compared to the full-lengthnative protein, e.g. its specific antigenic property. “Variants” ofproteins or peptides as defined in the context of the present inventionmay comprise conservative amino acid substitution(s) compared to theirnative, i.e. non-mutated physiological, sequence. Those amino acidsequences as well as their encoding nucleotide sequences in particularfall under the term variants as defined herein. Substitutions in whichamino acids, which originate from the same class, are exchanged for oneanother are called conservative substitutions. In particular, these areamino acids having aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or amino acids,the side chains of which can enter into hydrogen bridges, e.g. sidechains which have a hydroxyl function. This means that e.g. an aminoacid having a polar side chain is replaced by another amino acid havinga likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret al. (ed.), Elsevier, Amsterdam). A “variant” of a protein or peptidemay have at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% amino acididentity over a stretch of 10, 20, 30, 50, 75 or 100 amino acids of suchprotein or peptide. Furthermore, variants of proteins or peptides asdefined herein, which may be encoded by a nucleic acid molecule, mayalso comprise those sequences, wherein nucleotides of the encodingnucleic acid sequence are exchanged according to the degeneration of thegenetic code, without leading to an alteration of the respective aminoacid sequence of the protein or peptide, i.e. the amino acid sequence orat least part thereof may not differ from the original sequence withinthe above meaning. Preferably, a variant of a protein comprises afunctional variant of the protein, which means that the variant exertsthe same effect or functionality as the protein it is derived from.

Vector: The term “vector” refers to a nucleic acid molecule, preferablyto an artificial nucleic acid molecule. A vector in the context of thepresent invention is suitable for incorporating or harboring a desirednucleic acid sequence, such as a nucleic acid sequence comprising acoding sequence. Such vectors may be storage vectors, expressionvectors, cloning vectors, transfer vectors etc. A storage vector is avector which allows the convenient storage of a nucleic acid molecule,for example, of an mRNA molecule. Thus, the vector may comprise asequence corresponding, e.g., to a desired mRNA sequence or a partthereof, such as a sequence corresponding to the coding sequence and the3′-UTR and/or the 5′-UTR of an mRNA. An expression vector may be usedfor production of expression products such as RNA, e.g. mRNA, orpeptides, polypeptides or proteins. For example, an expression vectormay comprise sequences needed for transcription of a sequence stretch ofthe vector, such as a promoter sequence, e.g. an RNA polymerase promotersequence. A cloning vector is typically a vector that contains a cloningsite, which may be used to incorporate nucleic acid sequences into thevector. A cloning vector may be, e.g., a plasmid vector or abacteriophage vector. A transfer vector may be a vector which issuitable for transferring nucleic acid molecules into cells ororganisms, for example, viral vectors. A vector in the context of thepresent invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. Preferably, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector.

Vehicle: A vehicle is typically understood to be a material that issuitable for storing, transporting, and/or administering a compound,such as a pharmaceutically active compound. For example, it may be aphysiologically acceptable liquid which is suitable for storing,transporting, and/or administering a pharmaceutically active compound.

3′-untranslated region (3′-UTR): Generally, the term “3”-UTR″ refers toa part of a nucleic acid molecule, which is located 3′ (i.e.“downstream”) of a coding sequence and which is not translated intoprotein. Typically, a 3′-UTR is the part of an mRNA which is locatedbetween the protein coding sequence (coding region or coding sequence(CDS)) and the poly(A) sequence of the mRNA. In the context of theinvention, the term 3′-UTR may also comprise elements, which are notencoded in the template, from which an RNA is transcribed, but which areadded after transcription during maturation, e.g. a poly(A) sequence. A3′-UTR of the mRNA is not translated into an amino acid sequence. The3′-UTR sequence is generally encoded by the gene which is transcribedinto the respective mRNA during the gene expression process. The genomicsequence is first transcribed into pre-mature mRNA, which comprisesoptional introns. The pre-mature mRNA is then further processed intomature mRNA in a maturation process. This maturation process comprisesthe steps of 5′-capping, splicing the pre-mature mRNA to excize optionalintrons and modifications of the 3′-end, such as polyadenylation of the3′-end of the pre-mature mRNA and optional endo-/or exonucleasecleavages etc. In the context of the present invention, a 3′-UTRcorresponds to the sequence of a mature mRNA which is located betweenthe stop codon of the protein coding sequence, preferably immediately 3′to the stop codon of the protein coding sequence, and the poly(A)sequence of the mRNA. The term “corresponds to” means that the 3′-UTRsequence may be an RNA sequence, such as in the mRNA sequence used fordefining the 3′-UTR sequence, or a DNA sequence which corresponds tosuch RNA sequence. In the context of the present invention, the term “a3′-UTR of a gene”, is the sequence which corresponds to the 3′-UTR ofthe mature mRNA derived from this gene, i.e. the mRNA obtained bytranscription of the gene and maturation of the pre-mature mRNA. Theterm “3′-UTR of a gene” encompasses the DNA sequence and the RNAsequence (both sense and antisense strand and both mature and immature)of the 3′-UTR. Preferably, the 3′-UTRs have a length of more than 20,30, 40 or 50 nucleotides.

5′-untranslated region (5′-UTR): Generally, the term “5”-UTR″ refers toa part of a nucleic acid molecule, which is located 5′ (i.e. “upstream”)of a coding sequence and which is not translated into protein. A 5′-UTRis typically understood to be a particular section of messenger RNA(mRNA), which is located 5′ of the coding sequence of the mRNA.Typically, the 5′-UTR starts with the transcriptional start site andends one nucleotide before the start codon of the coding sequence.Preferably, the 5′-UTRs have a length of more than 20, 30, 40 or 50nucleotides. The 5′-UTR may comprise elements for controlling geneexpression, also called regulatory elements. Such regulatory elementsmay be, for example, ribosomal binding sites. The 5′-UTR may bepost-transcriptionally modified, for example by addition of a 5′-cap. A5′-UTR of the mRNA is not translated into an amino acid sequence. The5′-UTR sequence is generally encoded by the gene which is transcribedinto the respective mRNA during the gene expression process. The genomicsequence is first transcribed into pre-mature mRNA, which comprisesoptional introns. The pre-mature mRNA is then further processed intomature mRNA in a maturation process. This maturation process comprisesthe steps of 5′-capping, splicing the pre-mature mRNA to excise optionalintrons and modifications of the 3′-end, such as polyadenylation of the3′-end of the pre-mature mRNA and optional endo-/or exonucleasecleavages etc. In the context of the present invention, a 5′-UTRcorresponds to the sequence of a mature mRNA which is located betweenthe start codon and, for example, the 5′-cap. Preferably, the 5′-UTRcorresponds to the sequence which extends from a nucleotide located 3′to the 5′-cap, more preferably from the nucleotide located immediately3′ to the 5′-cap, to a nucleotide located 5′ to the start codon of theprotein coding sequence, preferably to the nucleotide locatedimmediately 5′ to the start codon of the protein coding sequence. Thenucleotide located immediately 3′ to the 5′-cap of a mature mRNAtypically corresponds to the transcriptional start site. The term“corresponds to” means that the 5′-UTR sequence may be an RNA sequence,such as in the mRNA sequence used for defining the 5′-UTR sequence, or aDNA sequence which corresponds to such RNA sequence. In the context ofthe present invention, the term “a 5′-UTR of a gene” is the sequencewhich corresponds to the 5′-UTR of the mature mRNA derived from thisgene, i.e. the mRNA obtained by transcription of the gene and maturationof the pre-mature mRNA. The term “5′-UTR of a gene” encompasses the DNAsequence and the RNA sequence (both sense and antisense strand and bothmature and immature) of the 5′-UTR.

5′-terminal oligopyrimidine tract (TOP): The 5′-terminal oligopyrimidinetract (TOP) is typically a stretch of pyrimidine nucleotides located inthe 5′ terminal region of a nucleic acid molecule, such as the 5′terminal region of certain mRNA molecules or the 5′ terminal region of afunctional entity, e.g. the transcribed region, of certain genes. Thesequence starts with a cytidine, which usually corresponds to thetranscriptional start site, and is followed by a stretch of usuallyabout 3 to 30 pyrimidine nucleotides. For example, the TOP may comprise3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30 or even more nucleotides. The pyrimidinestretch and thus the 5′ TOP ends one nucleotide 5′ to the first purinenucleotide located downstream of the TOP. Messenger RNA that contains a5′ terminal oligopyrimidine tract is often referred to as TOP mRNA.Accordingly, genes that provide such messenger RNAs are referred to asTOP genes. TOP sequences have, for example, been found in genes andmRNAs encoding peptide elongation factors and ribosomal proteins.

TOP motif: In the context of the present invention, a TOP motif is anucleic acid sequence which corresponds to a 5′ TOP as defined above.Thus, a TOP motif in the context of the present invention is preferablya stretch of pyrimidine nucleotides having a length of 3-30 nucleotides.Preferably, the TOP-motif consists of at least 3 pyrimidine nucleotides,preferably at least 4 pyrimidine nucleotides, preferably at least 5pyrimidine nucleotides, more preferably at least 6 nucleotides, morepreferably at least 7 nucleotides, most preferably at least 8 pyrimidinenucleotides, wherein the stretch of pyrimidine nucleotides preferablystarts at its 5′-end with a cytosine nucleotide. In TOP genes and TOPmRNAs, the TOP-motif preferably starts at its 5′-end with thetranscriptional start site and ends one nucleotide 5′ to the first purinresidue in said gene or mRNA. A TOP motif in the sense of the presentinvention is preferably located at the 5′-end of a sequence whichrepresents a 5′-UTR or at the 5′-end of a sequence which codes for a5′-UTR. Thus, preferably, a stretch of 3 or more pyrimidine nucleotidesis called “TOP motif” in the sense of the present invention if thisstretch is located at the 5′-end of a respective sequence, such as theartificial nucleic acid molecule, the 5′-UTR element of the artificialnucleic acid molecule, or the nucleic acid sequence which is derivedfrom the 5′-UTR of a TOP gene as described herein. In other words, astretch of 3 or more pyrimidine nucleotides, which is not located at the5′-end of a 5′-UTR or a 5′-UTR element but anywhere within a 5′-UTR or a5′-UTR element, is preferably not referred to as “TOP motif”.

TOP gene: TOP genes are typically characterised by the presence of a 5′terminal oligopyrimidine tract. Furthermore, most TOP genes arecharacterized by a growth-associated translational regulation. However,also TOP genes with a tissue specific translational regulation areknown. As defined above, the 5′-UTR of a TOP gene corresponds to thesequence of a 5′-UTR of a mature mRNA derived from a TOP gene, whichpreferably extends from the nucleotide located 3′ to the 5′-cap to thenucleotide located 5′ to the start codon. A 5′-UTR of a TOP genetypically does not comprise any start codons, preferably no upstreamAUGs (uAUGs) or upstream coding sequences (uORFs). Therein, upstreamAUGs and upstream coding sequences are typically understood to be AUGsand coding sequences that occur 5′ of the start codon (AUG) of thecoding sequence that should be translated. The 5′-UTRs of TOP genes aregenerally rather short. The lengths of 5′-UTRs of TOP genes may varybetween 20 nucleotides up to 500 nucleotides, and are typically lessthan about 200 nucleotides, preferably less than about 150 nucleotides,more preferably less than about 100 nucleotides. Exemplary 5′-UTRs ofTOP genes in the sense of the present invention are the nucleic acidsequences extending from the nucleotide at position 5 to the nucleotidelocated immediately 5′ to the start codon (e.g. the ATG) in thesequences according to SEQ ID NOs: 1-1363 of the patent applicationWO2013/143700, whose disclosure is incorporated herewith by reference.In this context a particularly preferred fragment of a 5′-UTR of a TOPgene is a 5′-UTR of a TOP gene lacking the 5′TOP motif. The terms“5′-UTR of a TOP gene” or “5′-TOP UTR” preferably refer to the 5′-UTR ofa naturally occurring TOP gene.

Short Description of the Invention

The present invention is based on the surprising finding that at leastone Henipavirus peptide or protein, particularly at least one Hendravirus and/or Nipah virus peptide or protein encoded by an artificialnucleic acid can efficiently be expressed in a mammalian cell. Furtherunexpectedly, the artificial nucleic acid, e.g. an mRNA sequence of theinvention, is suitable for eliciting an immune response against Hendravirus and/or Nipah virus in a mammalian subject, in particular, in ahuman subject. The artificial nucleic acid invention, the compositioncomprising said artificial nucleic acid, and the vaccine induces veryefficiently antigen-specific immune responses against the encodedantigenic peptide or protein. Moreover, the artificial nucleic acidinvention, the composition comprising said artificial nucleic acid, andthe vaccine can be stored without cold chain (lyophilizable) enablingrapid and scalable vaccine production which is of major importance inthe context of pandemic Hendra virus and/or Nipah virus outbreaks.

In a first aspect, the present invention relates to artificial nucleicacids comprising at least one coding sequence encoding at least oneantigenic peptide or protein derived from a Henipavirus or a fragment orvariant thereof, wherein the at least one antigenic peptide or proteincomprises or consists of a Henipavirus RNA-directed RNA polymerase (L),Henipavirus fusion protein (F), Henipavirus non-structural protein (V),Henipavirus glycoprotein (G), Henipavirus nucleoprotein (N), Henipavirusmatrix protein (M), Henipavirus phosphoprotein (P), Henipavirus proteinC, and Henipavirus protein W, or a fragment or variant of any of these.

In a preferred embodiment, the invention relates to an artificialnucleic acid comprising at least one coding sequence encoding at leastone antigenic peptide or protein derived from glycoprotein and/or fusionprotein of a Henipavirus or a fragment or variant thereof.

In a preferred embodiment, the Henipavirus is Nipah virus or Hendravirus.

In another preferred embodiment, the at least one antigenic peptide orprotein comprises or consists of a Hendra virus fusion protein, and/orHendra virus glycoprotein, and/or Nipah virus fusion protein and/orNipah virus glycoprotein, a fragment or variant of any of these.

The at least one antigenic peptide or protein, provided by theartificial nucleic acid, may additionally comprise an N-terminalheterologous signal peptide, preferably selected from an IgE-leader oran HA-A signal peptide, to improve secretion of the antigenic peptide orprotein.

The artificial nucleic acid may be monocistronic, bicistronic ormulticistronic.

The artificial nucleic acid sequence according to the invention may be amodified nucleic acid sequence.

The artificial nucleic acid may comprises an untranslated region (UTR),e.g. a 3′-UTR and/or 5′-UTR, preferably a heterologous 3′-UTR and/or5′-UTR, preferably derived from a gene encoding a stable mRNA.

In a preferred embodiment, the artificial nucleic acid is an RNA,preferably an mRNA, wherein the RNA is a stabilized RNA.

The artificial nucleic acid may further comprise a 5′-cap structure,and/or a 5′-UTR, and/or a Poly(A)sequence and/or a Poly(C) sequenceand/or a histone stem-loop, and/or a 3′-UTR.

In another aspect, the invention relates to a composition comprising atleast one artificial nucleic acid as described herein and at least onepharmaceutically acceptable carrier.

The composition may comprise a plurality or at least more than one ofthe artificial nucleic acids encoding a different antigenic peptide orprotein derived from a Henipavirus or from a homolog, fragment orvariant thereof, wherein the Henipavirus may be selected from Hendravirus and/or Nipah virus.

The artificial nucleic acid comprised in the composition mayadditionally be complexed with one or more cationic or polycationiccomponent, preferably with cationic or polycationic polymers, cationicor polycationic peptides or proteins, e.g. protamine, cationic orpolycationic lipids.

In an embodiment, the at least one artificial nucleic acid is complexedwith protamine.

In an embodiment, the at least one artificial nucleic acid of theinvention is complexed with a polymeric, preferably a polymer (e.g.peptide polymer) in conjunction with a lipidoid (e.g. 3-C12 OH).

The composition may comprise at least one protamine complexed artificialnucleic acid and at least one free artificial nucleic acid, wherein themolar ratio of the complexed nucleic acid to the free nucleic acid about1:1.

In another embodiment, the composition may comprise the artificialnucleic acid of the invention complexed with one or more lipids, therebyforming liposomes, lipid nanoparticles and/or lipoplexes.

The composition may further comprise at least one adjuvant component.

The present invention is also directed to the use of the artificialnucleic acid in treatment or prophylaxis of an infection withHenipavirus.

In particular, the present invention is directed to the use of theartificial nucleic acid in treatment or prophylaxis of an infection withHendra virus or a disorder related to such an infection.

Moreover, the present invention is directed to the use of the artificialnucleic acid in treatment or prophylaxis of an infection with Nipahvirus or a disorder related to such an infection.

The present invention also concerns a Henipavirus vaccine, in particulara Nipah virus vaccine and a Hendra virus vaccine.

The invention further concerns a method of treating or preventing adisorder or a disease in a mammalian subject or an avian subject, firstand second medical uses of the artificial nucleic acid, compositions andvaccines. Further, the invention is directed to a kit, particularly to akit of parts, comprising the artificial nucleic acid, compositions andvaccines.

DETAILED DESCRIPTION OF THE INVENTION

The present application is filed together with a sequence listing inelectronic format, which is part of the description of the presentapplication. The information contained in the electronic format of thesequence listing filed together with this application is incorporatedherein by reference in its entirety. Where reference is made herein to a“SEQ ID NO:” the corresponding nucleic acid sequence or amino acid (aa)sequence in the sequence listing having the respective identifier isreferred to. For many sequences, the sequence listing also providesdetailed information, e.g. regarding certain structural features,sequence optimizations, GenBank identifiers, or regarding its codingcapacity. In particular, such information may be provided under theidentifier <223> in the sequence listing. Accordingly, informationprovided under identifier <223> is explicitly included herein in itsentirety and has to be understood as part of the description of theinvention.

In a first aspect, the invention relates to an artificial nucleic acidcomprising at least one coding sequence encoding at least one antigenicpeptide or protein derived from a Henipavirus or a fragment or variantthereof.

Henipavirus:

In the context of the present invention, the term “Henipavirus”comprises any Henipavirus irrespective of genotype, species, strain,isolate, or serotype (NCBI taxonomy ID: 260964). Preferably, the termHenipavirus relates to a virus genus comprising virus strains selectedfrom Cedar henipavirus or Cedar virus (NCBI taxonomy ID: 1221391),Ghanaian bat henipavirus or Bat paramyxovirus (NCBI taxonomy ID:665603), Mojiang henipavirus or Mojiang virus (NCBI taxonomy ID:1474807), Hendra virus (NCBI taxonomy ID: 928303), Nipah virus (NCBItaxonomy ID: 121791).

Henipavirus Peptides or Proteins:

Henipavirus is a genus of negative sense single stranded RNA virusesbelonging to the Paramyxovirinae virus superfamily (NCBI Taxonomy ID:11158). The Henipavirus genome is about 18 kb in size, encoding for nineproteins, comprising RNA-directed RNA polymerase (L), fusion protein(F), non-structural protein (V), glycoprotein (G), nucleoprotein (N),matrix protein (M), phosphoprotein (P), protein C, and protein W.

In particular, the term “Henipavirus protein” as used herein comprisesor consists of an individual structural or non-structural Henipavirusprotein. A Henipavirus peptide or protein in the meaning of the presentinvention may be any full length protein or fragment derived fromHenipavirus RNA-directed RNA polymerase (L), Henipavirus fusion protein(F), Henipavirus non-structural protein (V), Henipavirus glycoprotein(G), Henipavirus nucleoprotein (N), Henipavirus matrix protein (M),Henipavirus phosphoprotein (P), Henipavirus protein C, and Henipavirusprotein W.

Accordingly, the term “Henipavirus protein” as used in the presentinvention may relate to an amino acid sequence corresponding to anyHenipavirus RNA-directed RNA polymerase (L), Henipavirus fusion protein(F), Henipavirus non-structural protein (V), Henipavirus glycoprotein(G), Henipavirus nucleoprotein (N), Henipavirus matrix protein (M),Henipavirus phosphoprotein (P), Henipavirus protein C, and Henipavirusprotein W.

The term “Henipavirus antigenic peptide or protein” as used in thepresent invention may relate to an amino acid sequence corresponding toany Henipavirus RNA-directed RNA polymerase (L), Henipavirus fusionprotein (F), Henipavirus non-structural protein (V), Henipavirusglycoprotein (G), Henipavirus nucleoprotein (N), Henipavirus matrixprotein (M), Henipavirus phosphoprotein (P), Henipavirus protein C, andHenipavirus protein W capable. Any Henipavirus peptide or proteinprovided herein, or any a fragment or variant thereof, can cause animmune response when administered to a subject. Therefore, allHenipavirus proteins or peptides provided herein can be considered asantigens in the context of the present invention.

Any Henipavirus peptide or protein provided herein, or any a fragment orvariant thereof, can cause an immune response when administered to asubject. Therefore, all Henipavirus proteins or peptides provided hereincan be considered as antigens in the context of the present invention.

In an embodiment, the Henipavirus peptide or protein as defined above isselected from Cedar henipavirus or Cedar virus (NCBI taxonomy ID:1221391), Ghanaian bat henipavirus or Bat paramyxovirus (NCBI taxonomyID: 665603), Mojiang henipavirus or Mojiang virus (NCBI taxonomy ID:1474807), Hendra virus (NCBI taxonomy ID: 928303), Nipah virus (NCBItaxonomy ID: 121791).

Accordingly, in preferred embodiments, the artificial nucleic acid asdefined herein, comprising at least one coding sequence encoding atleast one antigenic peptide or protein, wherein the at least oneantigenic peptide or protein comprises or consists of a RNA-directed RNApolymerase (L), fusion protein, non-structural protein, glycoprotein,nucleoprotein, matrix protein, phosphoprotein, protein C, and protein W,or a fragment or variant of any of these.

In embodiments, the Henipavirus is selected from Hendra virus (NCBItaxonomy ID: 928303) and Nipah virus (NCBI taxonomy ID: 121791).

Accordingly, in a preferred embodiment, the Henipavirus peptide orprotein is selected from a Hendra virus peptide or protein or Nipahvirus peptide or protein.

More preferably, the at least one antigenic peptide or protein comprisesor consists of a Hendra virus fusion protein, and/or Hendra virusglycoprotein, and/or Nipah virus fusion protein and/or Nipah virusglycoprotein, a fragment or variant of any of these.

In preferred embodiments, the at least one encoded antigenic peptide orprotein comprises at least one of the amino acid sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one ofSEQ ID NOs: 1-26, 573-598, 807-832, 1041-1066, 1513-1515 or provided inTable 1, Table 1B, Table 2, and Table 2B, or a fragment or variant ororthologue or paralogue of any of these.

Hendra Virus Peptides or Proteins:

The term “Hendra virus protein” or “Hendra virus protein” as used in thepresent invention may relate to an amino acid sequence corresponding toany Hendra virus RNA-directed RNA polymerase (L), Hendra virus fusionprotein (F), Hendra virus non-structural protein (V), Hendra virusglycoprotein (G), Hendra virus nucleoprotein (N), Hendra virus matrixprotein (M), Hendra virus phosphoprotein (P), Hendra virus protein C,and Hendra virus protein W.

Any Hendra virus peptide or protein provided herein, or any a fragmentor variant thereof, can cause an immune response when administered to asubject. Therefore, all Hendra virus proteins or peptides providedherein can be considered as antigens in the context of the presentinvention.

In some embodiments described herein, the at least one Hendra virusantigenic peptide or protein encoded by the at least one coding sequenceof the artificial nucleic acid may consist of an individual Hendra virusprotein, the amino acid sequence of which does typically not comprise anN-terminal Methionine residue. It is thus understood that the phrase“artificial nucleic acid comprising at least one coding sequenceencoding at least antigenic peptide or protein derived from a Hendravirus . . . ” relates to a protein or peptide comprising the amino acidsequence of said Hendra virus protein and—if the amino acid sequence ofthe respective Hendra virus protein does not comprise such an N-terminalMethionine residue—an introduced N-terminal Methionine residue.

In the context of the present invention a fragment of a protein or avariant thereof encoded by the at least one coding sequence of theartificial nucleic acid according to the invention may typicallycomprise an amino acid sequence having a sequence identity of at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of atleast 70%, more preferably of at least 80%, even more preferably atleast 85%, even more preferably of at least 90% and most preferably ofat least 95% or even 97%, with an amino acid sequence of the respectivenaturally occurring full-length Hendra virus protein or a variantthereof, preferably as disclosed in Table 1 or Table 1B.

In a preferred embodiment, the at least one coding sequence of theartificial nucleic acid sequence according to the invention preferablyencodes Hendra virus proteins selected from the proteins provided inTable 1 or Table 1B, or a fragment or variant thereof. Any Hendra virusprotein provided in Table 1 or Table 1B, or any a fragment or variantthereof, can cause an immune response when administered to anindividual. Therefore, all Hendra virus proteins provided in Table 1 orTable 1B can be considered as preferred Hendra virus antigens in thecontext of the present invention.

It is further preferred that the at least one coding sequence of theartificial nucleic acid sequence of the present invention encodes aHendra virus protein or peptide, or a fragment or variant thereof,wherein the Hendra virus protein or peptide is an antigen selected fromthe antigens listed in Table 1. Therein, each row corresponds to aHendra virus antigenic peptide or protein as identified by therespective gene name (first column “Name”) and the NCBI databaseaccession number of the corresponding protein (second column “AccessionNo.”). The third column (“A”, “Protein”) in Table 1 indicates the SEQ IDNOs corresponding to the respective amino acid sequence as providedherein. The SEQ ID NOs corresponding to the nucleic acid sequence of thewild type nucleic acid sequence encoding the Hendra virus antigenicprotein or peptide is indicated in the fourth column (“B”, “CDS wt”).The following columns (“C”-“J”) provides the SEQ ID NOs corresponding tomodified nucleic acid sequences (opt1, opt2, opt3, opt4, opt5, opt6,opt7) of the nucleic acid sequences as described herein that encode theHendra virus protein or peptide preferably having the amino acidsequence as defined by the SEQ ID NOs indicated in the third column(“A”) or by the database entry indicated in the second column(“Accession No.”). Additional information regarding each of thesequences provided in Table 1 may also be derived from the sequencelisting, in particular from the details provided therein underidentifier <223>.

TABLE 1 List of Hendra virus antigens: A B C D E F G H J Name AccessionNo. Protein CDS wt opt1 opt2 opt3 opt4 opt5 opt6 opt7 F NP_047111.2 8 3460 86 112 138 164 190 216 F AEB21233.1 9 35 61 87 113 139 165 191 217 FAEQ38114.1 10 36 62 88 114 140 166 192 218 F AAB39505.1 11 37 63 89 115141 167 193 219 G NP_047112.2 19 45 71 97 123 149 175 201 227 GAEB21225.1 20 46 72 98 124 150 176 202 228 G AEB21216.1 21 47 73 99 125151 177 203 229 G AEB21206.1 22 48 74 100 126 152 178 204 230 GAEQ38052.1 23 49 75 101 127 153 179 205 231 G AEQ38115.1 24 50 76 102128 154 180 206 232 G AEQ38108.1 25 51 77 103 129 155 181 207 233 GAAV80426.1 26 52 78 104 130 156 182 208 234

According to preferred embodiments, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Hendra virus antigenic peptide or peptide as described herein,wherein the at least one Hendra virus antigenic peptide or proteincomprises at least one amino acid sequence being identical or at least50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 8-11,19-26, 580-583, 591-598, 814-817, 825-832, 1048-1051, 1059-1066 or afragment or variant or orthologue or paralogue of any of these.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Hendra virus antigenic peptide or peptide as described herein,wherein the at least one Hendra virus antigenic peptide or proteincomprises an amino acid sequence according to any one of SEQ ID NOs:8-11 and 19-26, or a homolog, fragment or variant of any of thesesequences (see Table 1, column “A”).

In an embodiment the Hendra virus antigenic peptide or protein isderived from a Hendra virus Fusion protein (F) according to SEQ ID NOs:8-11.

In this context it is further preferred that the at least one codingsequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Hendra virus peptide or protein which isderived from Hendra virus fusion protein (F), or a fragment or variantthereof, wherein the Hendra virus fusion protein (F) is selected fromthe Hendra virus fusion protein amino acid sequences listed in Table 1.

Therein, rows corresponding to a Hendra virus fusion protein (F) (SEQ IDNOs: 8-11) can be identified by the respective gene name (first column“Name”: “F”) and the database accession number of the correspondingprotein (second column “Accession No.”). The SEQ ID NOs corresponding tothe nucleic acid sequence of the wild type nucleic acid encoding theHendra virus fusion protein (F) or peptide is indicated in the fourthcolumn (“B”). The further columns (“C”-“J”) provide the SEQ ID NOscorresponding to modified nucleic acid sequences of the nucleic acids asdescribed herein that encode the Hendra virus fusion protein (F) orpeptide preferably having the amino acid sequence as defined by the SEQID NOs: 8-11 or by the database entry indicated in the second column(“Accession No.”).

In an embodiment the Hendra virus antigenic peptide or protein isderived from a Hendra virus glycoprotein (G) according to SEQ ID NOs:19-26.

In this context it is further preferred that the at least one codingsequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Hendra virus peptide or protein which isderived from Hendra virus glycoprotein (G), or a fragment or variantthereof, wherein the Hendra virus glycoprotein (G) is selected from theHendra virus fusion protein amino acid sequences listed in Table 1.

Therein, rows corresponding to a Hendra virus glycoprotein (G) (SEQ IDNOs: 19-26) can be identified by the respective gene name (first column“Name”: “F”) and the database accession number of the correspondingprotein (second column “Accession No.”). The SEQ ID NOs corresponding tothe nucleic acid sequence of the wild type nucleic acid encoding theHendra virus glycoprotein (G) or peptide is indicated in the fourthcolumn (“B”). The further columns (“C”-“J”) provide the SEQ ID NOscorresponding to modified nucleic acid sequences of the nucleic acids asdescribed herein that encode the Hendra virus glycoprotein (G) orpeptide preferably having the amino acid sequence as defined by the SEQID NOs: 19-26 or by the database entry indicated in the second column(“Accession No.”).

In a specific embodiment, Hendra virus glycoprotein (G) (SEQ ID NOs:19-26) is N-terminally truncated to generate a soluble form of theprotein (solG). The N-terminal truncation has to be adapted in a waythat membrane-bound domains of the protein are removed. The membranetopology of a protein can be determined using prediction algorithms ascommonly known in the art (e.g. HMMTop (Tusnády and Simon (1998)Principles Governing Amino Acid Composition of Integral MembraneProteins: Applications to Topology Prediction.” J. Mol. Biol. 283,489-506) or TMHMM (Krogh et al. Predicting transmembrane proteintopology with a hidden Markov model: Application to complete genomes.Journal of Molecular Biology, 305(3):567-580, January 2001).

Hendra virus full length glycoprotein (G) polypeptides consist of 604amino acids (see Table 1).

In embodiments, soluble forms of the Hendra virus G protein (solG) aregenerated by truncating the first 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 amino acids of the protein according to SEQ ID NOs:19-26. In other words, amino acids 69-604, 70-604, 71-604, 72-604,73-604, 74-604, 75-604, 76-604, 77-604, 78-604, 79-604, 80-604, 81-604of the proteins according to SEQ ID NOs: 19-26 are soluble forms ofglycoprotein (solG).

In preferred embodiments, soluble forms of the protein (solG) aregenerated by truncating the first 70 amino acids of the proteinaccording to SEQ ID NOs: 19-26. In other words, amino acids 71-604 ofthe proteins according to SEQ ID NOs: 19-26 are soluble forms ofglycoprotein (solG).

In another specific embodiment, soluble forms of the protein (solG) aregenerated by truncating the first 73 amino acids of the proteinaccording to SEQ ID NOs: 19-26. In other words, amino acids 74-604 ofthe proteins according to SEQ ID NOs: 19-26 are soluble forms ofglycoprotein (solG).

In this context it is preferred that the at least one coding sequence ofthe artificial nucleic acid sequence of the present invention encodes atleast one Hendra virus antigenic peptide or protein which is derivedfrom Hendra virus soluble glycoprotein (solG) as defined above, or afragment or variant thereof. Suitable Hendra virus soluble glycoprotein(solG) proteins (and respective nucleic acid coding sequences) areprovided in Table 1B.

To facilitate secretion of the truncated solG protein, elements thatpromote secretion may be N-terminally fused to the (N-terminallytruncated) Hendra virus solG proteins as specified above. In specificembodiments, heterologous secretory signal peptides may be used,preferably selected from the list comprising SEQ ID NOs: 258-282,310-316 wherein IgE leader (SEQ ID NO: 264) and HA signal peptide (SEQID NO: 282) are particularly preferred. Further details about secretorysignal peptides are provided in the paragraph “Secretory signalpeptides” of the present application and in Table 3.

Accordingly, in this context it is preferred that the at least onecoding sequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Hendra virus antigenic peptide or proteinwhich is derived from a truncated Hendra virus solG as defined above, ora fragment or variant thereof, additionally comprising an N-terminalheterologous secretory signal peptide as defined above, or a fragment orvariant thereof. Suitable Hendra virus soluble glycoprotein (solG)proteins comprising an N-terminal heterologous secretory signal peptide(and respective nucleic acid coding sequences) are provided in Table 1B.

In a specific embodiment, Hendra virus fusion protein (F) (SEQ ID NOs:8-11) is N-terminally truncated to remove the endogenous secretorysignal peptide to generate truncated forms of Hendra virus fusionprotein (F). The N-terminal truncation has to be adapted in a way thatsecretory signal peptide of the protein are removed. Secretory signalpeptides of a protein can be determined using prediction algorithms ascommonly known in the art (e.g. SignalP (SignalP 4.0: discriminatingsignal peptides from transmembrane regions Thomas Nordahl Petersen,Søren Brunak, Gunnar von Heijne & Henrik Nielsen, Nature Methods,8:785-786, 2011).

Hendra virus full length Fusion (F) proteins commonly consist of 546amino acids (see Table 1).

In embodiments, Hendra F proteins lacking the endogenous signal peptide(SS) are generated by truncating the first 25 amino acids of the proteinaccording to SEQ ID NOs: 8-11. In other words, amino acids 26-546 of theproteins according to SEQ ID NOs: 8-11 are truncated forms ofglycoprotein (FdeISS).

In embodiments, Hendra F proteins lacking the endogenous signal peptide(SS) are generated by truncating the first 26 amino acids of the proteinaccording to SEQ ID NOs: 8-11. In other words, amino acids 27-546 of theproteins according to SEQ ID NOs: 8-11 are truncated forms of F protein(FdeISS).

In this context it is preferred that the at least one coding sequence ofthe artificial nucleic acid sequence of the present invention encodes atleast one Hendra virus antigenic peptide or protein which is derivedfrom Hendra virus FdeISS as defined above, or a fragment or variantthereof. Suitable Hendra virus truncated forms of Hendra F protein(FdeISS) (and respective nucleic acid coding sequences) are provided inTable 1B.

To facilitate secretion or improve secretion of the truncated F proteins(FdeISS), elements that promote secretion may be N-terminally fused tothe (N-terminally truncated) Hendra virus FdeISS proteins as specifiedabove. In specific embodiments, heterologous secretory signal peptidesmay be used, preferably selected from the list comprising SEQ ID NOs:258-282, 310-316, wherein IgE leader (SEQ ID NO: 264) and HA signalpeptide (SEQ ID NO: 282) are particularly preferred. Further detailsabout secretory signal peptides are provided in the paragraph “Secretorysignal peptides” of the present application and in Table 3.

Accordingly it is preferred that the at least one coding sequence of theartificial nucleic acid sequence of the present invention encodes atleast one Hendra virus antigenic peptide or protein which is derivedfrom a truncated Hendra virus FdeISS as defined above, or a fragment orvariant thereof, additionally comprising an N-terminal heterologoussecretory signal peptide as defined above, or a fragment or variantthereof. Suitable Hendra virus FdeISS proteins comprising an N-terminalheterologous secretory signal peptide (and respective nucleic acidcoding sequences) are provided in Table 1B.

In this context, it is particularly preferred that the at least onecoding sequence of the artificial nucleic acid sequence of the presentinvention encodes a Hendra virus FdeISS proteins as provided in Table1B. Particularly preferred are Hendra virus FdeISS proteins additionallycomprising an N-terminal heterologous secretory signal peptide(ICE-leader or HA signal peptide), preferably selected from the antigenslisted in Table 1B. In addition, Table 1B provides suitable nucleic acidsequences encoding FdeISS and FdeISS proteins additionally comprising anN-terminal heterologous secretory signal peptides.

In Table 1B provided herein, each row corresponds to a Hendra virusantigenic peptide or protein as identified by the respective constructname (first column “Name”). The second column (“A”, “Protein”) in Table1B indicates the SEQ ID NOs corresponding to the respective amino acidsequence as provided herein. The SEQ ID NOs corresponding to the nucleicacid sequence of the wild type nucleic acid sequence encoding theindicated Hendra virus antigenic protein or peptide is indicated in thefourth column (“B”, “CDS wt”). The following columns (“C”-“J”) providesthe SEQ ID NOs corresponding to modified nucleic acid sequences (opt1,opt2, opt3, opt4, opt5, opt6, opt7) of the nucleic acid sequences asdescribed herein that encode the Hendra virus protein or peptidepreferably having the amino acid sequence as defined by the SEQ ID NOsindicated in the second column (“A”). Additional information regardingeach of the sequences provided in Table 1B may also be derived from thesequence listing, in particular from the details provided therein underidentifier <223>.

TABLE 1B List of truncated Hendra virus antigens and Signal-peptidefusion proteins: A B C D E F G H J Name Protein CDS wt CDS opt1 CDS opt2CDS opt3 CDS opt4 CDS opt5 CDS opt6 CDS opt7 F(27-546) (FdelSS) 580 606632 658 684 710 736 762 788 F(27-546) (FdelSS) 581 607 633 659 685 711737 763 789 F(26-546) (FdelSS) 582 608 634 660 686 712 738 764 790F(27-546) (FdelSS) 583 609 635 661 687 713 739 765 791HsIgE(1-18)_F(27-546) 814 840 866 892 918 944 970 996 1022HsIgE(1-18)_F(27-546) 815 841 867 893 919 945 971 997 1023HsIgE(1-18)_F(26-546) 816 842 868 894 920 946 972 998 1024HsIgE(1-18)_F(27-546) 817 843 869 895 921 947 973 999 1025H1N1-HA(1-17)_F(27-546) 1048 1074 1100 1126 1152 1178 1204 1230 1256H1N1-HA(1-17)_F(27-546) 1049 1075 1101 1127 1153 1179 1205 1231 1257H1N1-HA(1-17)_F(26-546) 1050 1076 1102 1128 1154 1180 1206 1232 1258H1N1-HA(1-17)_F(27-546) 1051 1077 1103 1129 1155 1181 1207 1233 1259G(70-604) (solG) 591 617 643 669 695 721 747 773 799 G(70-604) (solG)592 618 644 670 696 722 748 774 800 G(70-604) (solG) 593 619 645 671 697723 749 775 801 G(70-604) (solG) 594 620 646 672 698 724 750 776 802G(70-604) (solG) 595 621 647 673 699 725 751 777 803 G(70-604) (solG)596 622 648 674 700 726 752 778 804 G(70-604) (solG) 597 623 649 675 701727 753 779 805 G(70-604) (solG) 598 624 650 676 702 728 754 780 806HsIgE(1-18)_G(70-604) 825 851 877 903 929 955 981 1007 1033HsIgE(1-18)_G(70-604) 826 852 878 904 930 956 982 1008 1034HsIgE(1-18)_G(70-604) 827 853 879 905 931 957 983 1009 1035HsIgE(1-18)_G(70-604) 828 854 880 906 932 958 984 1010 1036HsIgE(1-18)_G(70-604) 829 855 881 907 933 959 985 1011 1037HsIgE(1-18)_G(70-604) 830 856 882 908 934 960 986 1012 1038HsIgE(1-18)_G(70-604) 831 857 883 909 935 961 987 1013 1039HsIgE(1-18)_G(70-604) 832 858 884 910 936 962 988 1014 1040H1N1-HA(1-17)_G(70-604) 1059 1085 1111 1137 1163 1189 1215 1241 1267H1N1-HA(1-17)_G(70-604) 1060 1086 1112 1138 1164 1190 1216 1242 1268H1N1-HA(1-17)_G(70-604) 1061 1087 1113 1139 1165 1191 1217 1243 1269H1N1-HA(1-17)_G(70-604) 1062 1088 1114 1140 1166 1192 1218 1244 1270H1N1-HA(1-17)_G(70-604) 1063 1089 1115 1141 1167 1193 1219 1245 1271H1N1-HA(1-17)_G(70-604) 1064 1090 1116 1142 1168 1194 1220 1246 1272H1N1-HA(1-17)_G(70-604) 1065 1091 1117 1143 1169 1195 1221 1247 1273H1N1-HA(1-17)_G(70-604) 1066 1092 1118 1144 1170 1196 1222 1248 1274

According to preferred embodiments, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Hendra virus antigenic peptide or peptide as provided herein,wherein the at least one Hendra virus antigenic peptide or proteincomprises at least one amino acid sequence being identical or at least50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 580-583,591-598, 814-817, 825-832, 1048-1051, 1059-1066, or a fragment orvariant or orthologue or paralogue of any of these.

In other embodiments, the inventive artificial nucleic acid comprises orconsists of at least one coding sequence encoding at least one antigenicpeptide or protein derived from a Hendra virus RNA-directed RNApolymerase (L), Hendra virus fusion protein (F), Hendra virusnon-structural protein (V), Hendra virus glycoprotein (G), Hendra virusnucleoprotein (N), Hendra virus matrix protein (M), Hendra virusphosphoprotein (P), Hendra virus protein C, and Hendra virus protein Wor a fragment or variant of any of these.

In another embodiment, the inventive artificial nucleic acid comprisesor consists of at least one coding sequence encoding at least oneantigenic peptide or protein derived from Hendra virus RNA-directed RNApolymerase (L), or a fragment or variant thereof. In another embodiment,the inventive artificial nucleic acid comprises or consists of at leastone coding sequence encoding at least one antigenic peptide or proteinderived from Hendra virus non-structural protein (V), or a fragment orvariant thereof. In another embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one antigenic peptide or protein derived from Hendra virusnucleoprotein (N), or a fragment or variant thereof. In anotherembodiment, the inventive artificial nucleic acid comprises or consistsof at least one coding sequence encoding at least one antigenic peptideor protein derived from Hendra virus matrix protein (M), or a fragmentor variant thereof. In another embodiment, the inventive artificialnucleic acid comprises or consists of at least one coding sequenceencoding at least one antigenic peptide or protein derived from Hendravirus phosphoprotein (P), or a fragment or variant thereof. In anotherembodiment, the inventive artificial nucleic acid comprises or consistsof at least one coding sequence encoding at least one antigenic peptideor protein derived from Hendra virus protein C, or a fragment or variantthereof. In another embodiment, the inventive artificial nucleic acidcomprises or consists of at least one coding sequence encoding at leastone antigenic peptide or protein derived from Hendra virus protein W, ora fragment or variant thereof.

Nipah Virus Peptides or Proteins:

The term “Nipah virus protein” or “Nipah virus peptide” as used in thepresent invention may relate to an amino acid sequence corresponding toany Nipah virus RNA-directed RNA polymerase (L), Nipah virus fusionprotein (F), Nipah virus non-structural protein (V), Nipah virusglycoprotein (G), Nipah virus nucleoprotein (N), Nipah virus matrixprotein (M), Nipah virus phosphoprotein (P), Nipah virus protein C, andNipah virus protein W.

Any Nipah virus peptide or protein provided herein, or any a fragment orvariant thereof, can cause an immune response when administered to asubject. Therefore, all Nipah virus proteins or peptides provided hereincan be considered as antigens in the context of the present invention.

In some embodiments described herein, the at least one Nipah virusantigenic peptide or protein encoded by the at least one coding sequenceof the artificial nucleic acid may consist of an individual Nipah virusprotein, the amino acid sequence of which does typically not comprise anN-terminal Methionine residue. It is thus understood that the phrase“artificial nucleic acid comprising at least one coding sequenceencoding at least one antigenic peptide or protein derived from a Nipahvirus . . . ” relates to a protein or peptide comprising the amino acidsequence of said Nipah virus protein and—if the amino acid sequence ofthe respective Nipah virus protein does not comprise such an N-terminalMethionine residue—an introduced N-terminal Methionine residue.

In the context of the present invention a fragment of a protein or avariant thereof encoded by the at least one coding sequence of theartificial nucleic acid according to the invention may typicallycomprise an amino acid sequence having a sequence identity of at least5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of atleast 70%, more preferably of at least 80%, even more preferably atleast 85%, even more preferably of at least 90% and most preferably ofat least 95% or even 97%, with an amino acid sequence of the respectivenaturally occurring full-length Nipah virus protein or a variantthereof, preferably as disclosed in Table 2 or Table 2B.

In a preferred embodiment, the at least one coding sequence of theartificial nucleic acid sequence according to the invention preferablyencodes Nipah virus proteins selected from the proteins provided inTable 2 or Table 2B, or a fragment or variant thereof. Any Nipah virusprotein provided in Table 2 or Table 2B, or any a fragment or variantthereof, can cause an immune response when administered to anindividual. Therefore, all Nipah virus proteins provided in Table 2 orTable 2B can be considered as preferred Nipah virus antigens in thecontext of the present invention.

It is further preferred that the at least one coding sequence of theartificial nucleic acid sequence of the present invention encodes aNipah virus protein or peptide, or a fragment or variant thereof,wherein the Nipah virus protein or peptide is an antigen selected fromthe antigens listed in Table 2. Therein, each row corresponds to a Nipahvirus antigenic peptide or protein as identified by the respective genename (first column “Name”) and the NCBI database accession number of thecorresponding protein (second column “Accession No.”). The third column(“A”, “protein”) in Table 2 indicates the SEQ ID NOs corresponding tothe respective amino acid sequence as provided herein. The SEQ ID NOscorresponding to the nucleic acid sequence of the wild type nucleic acidsequence encoding the Nipah virus antigenic protein or peptide isindicated in the fourth column (“B”, “CDS wt”). The following columns(“C”-“J”) provides the SEQ ID NOs corresponding to modified nucleic acidsequences (opt1, opt2, opt3, opt4, opt5, opt6, opt7) of the nucleic acidsequences as described herein that encode the Nipah virus protein orpeptide preferably having the amino acid sequence as defined by the SEQID NOs indicated in the third column (“A”) or by the database entryindicated in the second column (“Accession No.”). Additional informationregarding each of the sequences provided in Table 2 may also be derivedfrom the sequence listing, in particular from the details providedtherein under identifier <223>.

TABLE 2 List of Nipah virus antigens: A B C D E F G H J Name AccessionNo. Protein CDS wt opt1 opt2 opt3 opt4 opt5 opt6 opt7 F AAK50553.1 1 2753 79 105 131 157 183 209 F AEZ01388.1 2 28 54 80 106 132 158 184 210 FAAY43915.1 3 29 55 81 107 133 159 185 211 F CAF25496.1 4 30 56 82 108134 160 186 212 F AAM13405.1 5 31 57 83 109 135 161 187 213 F CBM41033.16 32 58 84 110 136 162 188 214 F AEZ01396.1 7 33 59 85 111 137 163 189215 G AAK50554.1 12 38 64 90 116 142 168 194 220 G AEZ01389.1 13 39 6591 117 143 169 195 221 G ACT32615.1 14 40 66 92 118 144 170 196 222 GCAF25497.1 15 41 67 93 119 145 171 197 223 G CBM41034.1 16 42 68 94 120146 172 198 224 G AAX51853.1 17 43 69 95 121 147 173 199 225 GAEZ01397.1 18 44 70 96 122 148 174 200 226

According to preferred embodiments, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Nipah virus antigenic peptide or peptide as described herein,wherein the at least one Nipah virus antigenic peptide or proteincomprises at least one amino acid sequence being identical or at least50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 1-7,12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047, 1052-1058,1513-1515 or a fragment or variant or orthologue or paralogue of any ofthese.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Nipah virus antigenic peptide or peptide as described herein,wherein the at least one Nipah virus antigenic peptide or proteincomprises an amino acid sequence according to any one of SEQ ID NOs: 1-7and 12-18, or a homolog, fragment or variant of any of these sequences(see Table 2, column “A”).

In an embodiment the Nipah virus antigenic peptide or protein is derivedfrom a Nipah virus Fusion protein (F) according to SEQ ID NOs: 1-7.

In this context it is further preferred that the at least one codingsequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Nipah virus peptide or protein which isderived from Nipah virus fusion protein (F), or a fragment or variantthereof, wherein the Nipah virus fusion protein (F) is selected from theNipah virus fusion protein amino acid sequences listed in Table 2.

Therein, rows corresponding to a Nipah virus fusion protein (F) (SEQ IDNOs: 1-7) can be identified by the respective gene name (first column“Name”: “F”) and the database accession number of the correspondingprotein (second column “Accession No.”). The SEQ ID NOs: correspondingto the nucleic acid sequence of the wild type nucleic acid encoding theNipah virus fusion protein (F) or peptide is indicated in the fourthcolumn (“B”). The further columns (“C”-“J”) provide the SEQ ID NOscorresponding to modified nucleic acid sequences of the nucleic acids asdescribed herein that encode the Nipah virus fusion protein (F) orpeptide preferably having the amino acid sequence as defined by the SEQID NOs: 1-7 or by the database entry indicated in the second column(“Accession No.”).

In an embodiment the Nipah virus antigenic peptide or protein is derivedfrom a Nipah virus glycoprotein (G) according to SEQ ID NOs: 12-18.

In this context it is further preferred that the at least one codingsequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Nipah virus peptide or protein which isderived from Nipah virus glycoprotein (G), or a fragment or variantthereof, wherein the Nipah virus glycoprotein (G) is selected from theNipah virus fusion protein amino acid sequences listed in Table 2.

Therein, rows corresponding to a Nipah virus glycoprotein (G) (SEQ IDNOs: 12-18) can be identified by the respective gene name (first column“Name”: “F”) and the database accession number of the correspondingprotein (second column “Accession No.”). The SEQ ID NOs: correspondingto the nucleic acid sequence of the wild type nucleic acid encoding theNipah virus glycoprotein (G) or peptide is indicated in the fourthcolumn (“B”). The further columns (“C”-“J”) provide the SEQ ID NOscorresponding to modified nucleic acid sequences of the nucleic acids asdescribed herein that encode the Nipah virus glycoprotein (G) or peptidepreferably having the amino acid sequence as defined by the SEQ ID NOs:12-18 or by the database entry indicated in the second column(“Accession No.”).

In a specific embodiment, Nipah virus glycoprotein (G) (SEQ ID NOs:12-18) is N-terminally truncated to generate a soluble form of theprotein (solG). The N-terminal truncation has to be adapted in a waythat membrane-bound domains of the protein are removed. The membranetopology of a protein can be determined using prediction algorithms ascommonly known in the art.

Nipah virus glycoprotein (G) polypeptides commonly consist of 602 aminoacids (see Table 2).

In embodiments, soluble forms of the Nipah virus G protein (solG) aregenerated by truncating the first 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, or 80 amino acids of the protein according to SEQ ID NOs:12-18. In other words, amino acids 69-602, 70-602, 71-602, 72-602,73-602, 74-602, 75-602, 76-602, 77-602, 78-602, 79-602, 80-602, 81-602of the proteins according to SEQ ID NOs: 12-18 are soluble forms ofglycoprotein (solG).

In a preferred embodiments, soluble forms of the Nipah virus protein(solG) are generated by truncating the first 70 amino acids of theprotein according to SEQ ID NOs: 12-18. In other words, amino acids71-602 of the proteins according to SEQ ID NOs: 12-18 are soluble formsof glycoprotein (solG).

In another specific embodiment, soluble forms of the Nipah virus protein(solG) are generated by truncating the first 72 amino acids of theprotein according to SEQ ID NOs 12-18. In other words, amino acids73-602 of the proteins according to SEQ ID NOs 12-18 are soluble formsof glycoprotein (solG).

In this context it is preferred that the at least one coding sequence ofthe artificial nucleic acid sequence of the present invention encodes atleast one Nipah virus antigenic peptide or protein which is derived fromNipah virus soluble glycoprotein (solG) as defined above, or a fragmentor variant thereof. Suitable Nipah virus soluble glycoprotein (solG)proteins (and respective nucleic acid coding sequences) are provided inTable 2B.

To facilitate secretion of the truncated protein, elements that promotesecretion may be N-terminally fused to the (N-terminally truncated)Nipah virus solG proteins. In specific embodiments, secretory signalpeptides may be used, preferably selected from the list comprising SEQID NOs: 258-282, 310-316, wherein IgE leader (SEQ ID NO: 264) and HAsignal peptide (SEQ ID NO: 282) are particularly preferred. Furtherdetails about secretory signal peptides are provided in the paragraph“Secretory signal peptides” of the present application and in Table 3.

Accordingly, in this context it is preferred that the at least onecoding sequence of the artificial nucleic acid sequence of the presentinvention encodes at least one Hendra virus antigenic peptide or proteinwhich is derived from a truncated Nipah virus solG as defined above, ora fragment or variant thereof, additionally comprising an N-terminalheterologous secretory signal peptide as defined above, or a fragment orvariant thereof. Suitable Nipah virus soluble glycoprotein (solG)proteins comprising an N-terminal heterologous secretory signal peptide(and respective nucleic acid coding sequences) are provided in Table 2B.

In a specific embodiment, Nipah virus fusion protein (F) (SEQ ID NOs:1-7) is N-terminally truncated to remove the endogenous secretory signalpeptide to generate truncated forms of Nipah virus fusion protein (F).The N-terminal truncation has to be adapted in a way that secretorysignal peptide of the protein are removed. Secretory signal peptides ofa protein can be determined using prediction algorithms as commonlyknown in the art (e.g. SignalP (SignalP 4.0: discriminating signalpeptides from transmembrane regions Thomas Nordahl Petersen, SørenBrunak, Gunnar von Heijne & Henrik Nielsen, Nature Methods, 8:785-786,2011).

Nipah virus full length Fusion (F) proteins commonly consist of 546amino acids (see Table 2).

In embodiments, Nipah F proteins lacking the endogenous signal peptide(SS) are generated by truncating the first 25 amino acids of the proteinaccording to SEQ ID NOs: 1-7. In other words, amino acids 26-546 of theproteins according to SEQ ID NOs: 1-7 are truncated forms ofglycoprotein (FdeISS).

In embodiments, Nipah F proteins lacking the endogenous signal peptide(SS) are generated by truncating the first 26 amino acids of the proteinaccording to SEQ ID NOs: 1-7. In other words, amino acids 27-546 of theproteins according to SEQ ID NOs: 1-7 are truncated forms of F protein(FdeISS).

In this context it is preferred that the at least one coding sequence ofthe artificial nucleic acid sequence of the present invention encodes atleast one Nipah virus antigenic peptide or protein which is derived fromNipah virus FdeISS as defined above, or a fragment or variant thereof.Suitable truncated forms of Nipah F protein (FdeISS) (and respectivenucleic acid coding sequences) are provided in Table 2B.

To facilitate secretion or improve secretion of the truncated Nipah Fproteins (FdeISS), elements that promote secretion may be N-terminallyfused to the (N-terminally truncated) Nipah virus FdeISS proteins asspecified above. In specific embodiments, heterologous secretory signalpeptides may be used, preferably selected from the list comprising SEQID NOs: 258-282, 310-316, wherein IgE leader (SEQ ID NO: 264) and HAsignal peptide (SEQ ID NO: 282) are particularly preferred. Furtherdetails about secretory signal peptides are provided in the paragraph“Secretory signal peptides” of the present application and in Table 3.

Accordingly it is preferred that the at least one coding sequence of theartificial nucleic acid sequence of the present invention encodes atleast one antigenic peptide or protein which is derived from a truncatedNipah virus FdeISS as defined above, or a fragment or variant thereof,additionally comprising an N-terminal heterologous secretory signalpeptide as defined above, or a fragment or variant thereof. SuitableNipah virus FdeISS proteins comprising an N-terminal heterologoussecretory signal peptide (and respective nucleic acid coding sequences)are provided in Table 2B.

In this context, it is particularly preferred that the at least onecoding sequence of the artificial nucleic acid sequence of the presentinvention encodes a Nipah virus FdeISS proteins as provided in Table 2B.Particularly preferred are Nipah virus FdeISS proteins additionallycomprising an N-terminal heterologous secretory signal peptide(IGE-leader or HA signal peptide), preferably selected from the antigenslisted in Table 2B. In addition, Table 2B provides suitable nucleic acidsequences encoding FdeISS and FdeISS proteins additionally comprising anN-terminal heterologous secretory signal peptides.

In Table 2B provided herein, each row corresponds to a Nipah virusantigenic peptide or protein as identified by the respective constructname (first column “Name”). The second column (“A”, “Protein”) in Table2B indicates the SEQ ID NOs corresponding to the respective amino acidsequence as provided herein. The SEQ ID NOs corresponding to the nucleicacid sequence of the wild type nucleic acid sequence encoding theindicated Nipah virus antigenic protein or peptide is indicated in thefourth column (“B”, “CDS wt”). The following columns (“C”-“J”) providesthe SEQ ID NOs corresponding to modified nucleic acid sequences (opt1,opt2, opt3, opt4, opt5, opt6, opt7) of the nucleic acid sequences asdescribed herein that encode the Nipah virus protein or peptidepreferably having the amino acid sequence as defined by the SEQ ID NOsindicated in the second column (“A”). Additional information regardingeach of the sequences provided in Table 2B may also be derived from thesequence listing, in particular from the details provided therein underidentifier <223>.

TABLE 2B List of truncated Nipah virus antigens and Signal-peptidefusion proteins: A B C D E F G H J Name Protein CDS wt CDS opt1 CDS opt2CDS opt3 CDS opt4 CDS opt5 CDS opt6 CDS opt7 F(27-546) (FdelSS) 573 599625 651 677 703 729 755 781 F(27-546) (FdelSS) 574 600 626 652 678 704730 756 782 F(27-546) (FdelSS) 575 601 627 653 679 705 731 757 783F(27-546) (FdelSS) 576 602 628 654 680 706 732 758 784 F(27-546)(FdelSS) 577 603 629 655 681 707 733 759 785 F(27-546) (FdelSS) 578 604630 656 682 708 734 760 786 F(27-546) (FdelSS) 579 605 631 657 683 709735 761 787 HsIgE(1-18)_F(27-546) 807 833 859 885 911 937 963 989 1015HsIgE(1-18)_F(27-546) 808 834 860 886 912 938 964 990 1016HsIgE(1-18)_F(27-546) 809 835 861 887 913 939 965 991 1017HsIgE(1-18)_F(27-546) 810 836 862 888 914 940 966 992 1018HsIgE(1-18)_F(27-546) 811 837 863 889 915 941 967 993 1019HsIgE(1-18)_F(27-546) 812 838 864 890 916 942 968 994 1020HsIgE(1-18)_F(27-546) 813 839 865 891 917 943 969 995 1021H1N1-HA(1-17)_F(27-546) 1041 1067 1093 1119 1145 1171 1197 1223 1249H1N1-HA(1-17)_F(27-546) 1042 1068 1094 1120 1146 1172 1198 1224 1250H1N1-HA(1-17)_F(27-546) 1043 1069 1095 1121 1147 1173 1199 1225 1251H1N1-HA(1-17)_F(27-546) 1044 1070 1096 1122 1148 1174 1200 1226 1252H1N1-HA(1-17)_F(27-546) 1045 1071 1097 1123 1149 1175 1201 1227 1253H1N1-HA(1-17)_F(27-546) 1046 1072 1098 1124 1150 1176 1202 1228 1254H1N1-HA(1-17)_F(27-546) 1047 1073 1099 1125 1151 1177 1203 1229 1255G(70-602) (solG) 584 610 636 662 688 714 740 766 792 G(70-602) (solG)585 611 637 663 689 715 741 767 793 G(70-602) (solG) 586 612 638 664 690716 742 768 794 G(70-602) (solG) 587 613 639 665 691 717 743 769 795G(70-602) (solG) 588 614 640 666 692 718 744 770 796 G(70-602) (solG)589 615 641 667 693 719 745 771 797 G(70-602) (solG) 590 616 642 668 694720 746 772 798 HsIgE(1-18)_G(70-602) 818 844 870 896 922 948 974 10001026 HsIgE(1-18)_G(70-602) 819 845 871 897 923 949 975 1001 1027HsIgE(1-18)_G(70-602) 820 846 872 898 924 950 976 1002 1028HsIgE(1-18)_G(70-602) 821 847 873 899 925 951 977 1003 1029HsIgE(1-18)_G(70-602) 822 848 874 900 926 952 978 1004 1030HsIgE(1-18)_G(70-602) 823 849 875 901 927 953 979 1005 1031HsIgE(1-18)_G(70-602) 824 850 876 902 928 954 980 1006 1032H1N1-HA(1-17)_G(70-602) 1052 1078 1104 1130 1156 1182 1208 1234 1260H1N1-HA(1-17)_G(70-602) 1053 1079 1105 1131 1157 1183 1209 1235 1261H1N1-HA(1-17)_G(70-602) 1054 1080 1106 1132 1158 1184 1210 1236 1262H1N1-HA(1-17)_G(70-602) 1055 1081 1107 1133 1159 1185 1211 1237 1263H1N1-HA(1-17)_G(70-602) 1056 1082 1108 1134 1160 1186 1212 1238 1264H1N1-HA(1-17)_G(70-602) 1057 1083 1109 1135 1161 1187 1213 1239 1265H1N1-HA(1-17)_G(70-602) 1058 1084 1110 1136 1162 1188 1214 1240 1266HsSPARC(1-17)_F(27-546) 1513 1516 1519 1522 1525 1528 1531 1534 1537HsCTRB2(1-18)_F(27-546) 1514 1517 1520 1523 1526 1529 1532 1535 1538Nipah 1515 1518 1521 1524 1527 1530 1533 1536 1539henipavirus_AAK50553_F(1-26)_F(27-546)

According to preferred embodiments, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Nipah virus antigenic peptide or peptide as provided herein,wherein the at least one Nipah virus antigenic peptide or proteincomprises at least one amino acid sequence being identical or at least50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NOs: 573-579,584-590, 807-813, 818-824, 1041-1047, 1052-1058, 1513-1515 or a fragmentor variant or orthologue or paralogue of any of these.

In other embodiments, the inventive artificial nucleic acid comprises orconsists of at least one coding sequence encoding at least one antigenicpeptide or protein derived from a Nipah virus RNA-directed RNApolymerase (L), Nipah virus fusion protein (F), Nipah virusnon-structural protein (V), Nipah virus glycoprotein (G), Nipah virusnucleoprotein (N), Nipah virus matrix protein (M), Nipah virusphosphoprotein (P), Nipah virus protein C, and Nipah virus protein W ora fragment or variant of any of these.

In another embodiment, the inventive artificial nucleic acid comprisesor consists of at least one coding sequence encoding at least oneantigenic peptide or protein derived from Nipah virus RNA-directed RNApolymerase (L), or a fragment or variant thereof. In another embodiment,the inventive artificial nucleic acid comprises or consists of at leastone coding sequence encoding at least one antigenic peptide or proteinderived from Nipah virus non-structural protein (V), or a fragment orvariant thereof. In another embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one antigenic peptide or protein derived from Nipah virusnucleoprotein (N), or a fragment or variant thereof. In anotherembodiment, the inventive artificial nucleic acid comprises or consistsof at least one coding sequence encoding at least one antigenic peptideor protein derived from Nipah virus matrix protein (M), or a fragment orvariant thereof. In another preferred embodiment, the inventiveartificial nucleic acid comprises or consists of at least one codingsequence encoding at least one antigenic peptide or protein derived fromNipah virus phosphoprotein (P), or a fragment or variant thereof. Inanother embodiment, the inventive artificial nucleic acid comprises orconsists of at least one coding sequence encoding at least one antigenicpeptide or protein derived from Nipah virus protein C, or a fragment orvariant thereof. In another embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one antigenic peptide or protein derived from Nipah virus proteinW, or a fragment or variant thereof.

Additional Peptide or Protein Elements:

According to another particularly preferred embodiment, the artificialnucleic acid sequence, particularly the RNA sequence according to theinvention may additionally encode a further, preferably heterologouspeptide or protein elements, that e.g., promote secretion of the protein(secretory signal peptides), promote anchoring of the encoded antigen inthe plasma membrane (transmembrane domains), promote virus-like particleformation (VLP forming domains). In addition, the artificial nucleicacid sequence according to the present invention may additionally encodepeptide linker elements, self-cleaving peptides or helper peptides.Further, the artificial nucleic acid sequence according to the presentinvention may additionally encode an immunologic adjuvant sequence,and/or a dendritic cell targeting sequence.

Secretory Signal Peptides:

According another preferred embodiment, the artificial nucleic acidsequence, particularly the RNA sequence according to the invention mayadditionally or alternatively encode at least one secretory signalpeptide. Such signal peptides are sequences, which typically exhibit alength of about 15 to 30 amino acids and are preferably located at theN-terminus of the encoded peptide, without being limited thereto. Signalpeptides as defined herein preferably allow the transport of theHenipavirus and/or Hendra virus and/or Nipah virus antigenic peptide orproteins as encoded by the at least one artificial nucleic acid sequenceinto a defined cellular compartment, preferably the cell surface, theendoplasmic reticulum (ER) or the endosomal-lysosomal compartment.Examples of secretory signal peptide sequences as defined hereininclude, without being limited thereto, signal sequences of classical ornon-classical MHC-molecules (e.g. signal sequences of MHC I and IImolecules, e.g. of the MHC class I molecule HLA-A*0201), signalsequences of cytokines or immunoglobulins as defined herein, signalsequences of the invariant chain of immunoglobulins or antibodies asdefined herein, signal sequences of Lamp1, Tapasin, Erp57, Calretikulin,Calnexin, and further membrane associated proteins or of proteinsassociated with the endoplasmic reticulum (ER) or theendosomal-lysosomal compartment. Most preferably, signal sequences ofMHC class I molecule HLA-A*0201 may be used according to the presentinvention. For example, a signal peptide derived from HLA-A ispreferably used in order to promote secretion of the encoded Henipavirusand/or Hendra virus and/or Nipah virus antigen as defined herein or afragment or variant thereof. More preferably, an HLA-A signal peptide isfused to an encoded Henipavirus and/or Hendra virus and/or Nipah virusantigen as defined herein or to a fragment or variant thereof.Particularly preferred secretory signal peptides according to thepresent invention are provided in the sequence listing (SEQ ID NOs:258-282, 310-316). Further suitable secretory signal peptides may beselected from the list of amino acid sequences according to SEQ ID NOs:1-1115 and SEQ ID NO: 1728 of the patent application WO2017/081082, orfragments or variants of these sequences, herewith incorporated byreference. Particularly preferred secretory signal peptides in thecontext of the invention are IgE leader (HsIgE(1-18)) (SEQ ID NO: 264)HA signal peptide (H1N1-HA(1-17)) (SEQ ID NO: 282), HsSPARC (SEQ ID NO:281), and HsCTRB2 (SEQ ID NO: 267).

On nucleic acid level, particularly RNA level, any nucleotide sequencemoiety can be employed that encodes any of secretory signal peptidesused in the context of the present invention. Owing to the degeneratedgenetic code, e.g. in the case of most peptide sequences according toSEQ ID NOs: 258-282, 310-316 more than one particular nucleic acidsequence is conceivable as encoding the respective polypeptide. Whileeach and every such nucleic acid may generally be used in the context ofthe present invention, it is preferable that the nucleic acid sequencethat encodes the polypeptide sequence is selected such that its sequenceis optimized according to the general guidance provided in thisspecification.

In the context of the invention, it is particularly preferred that thesecretory signal peptide as defined herein is located at the N-terminusof the at least one antigenic peptide or protein, followed by aF-protein lacking its endogenous secretory signal peptide (FdeISS) asdefined above or followed by a G-protein lacking its endogenoustransmembrane domain (solG) as defined above (see also Table 1B andTable 2B).

In preferred embodiments, secretory signal peptides may suitably beselected from those provided in Table 3. In Table 3, each rowcorresponds to a secretory signal peptide as identified by therespective name (first column “Name”) and the Accession number (secondcolumn “NCBI Accession No.”) The third column (“A”, “Protein”) in Table3 indicates the SEQ ID NOs corresponding to the respective amino acidsequence as provided herein. The SEQ ID NOs corresponding to the nucleicacid sequence of the wild type nucleic acid sequence encoding theindicated secretory signal peptide is indicated in the fourth column(“B”, “CDS wt”). The following columns (“C-J”) provides the SEQ ID NOscorresponding to modified nucleic acid sequences (opt1, opt2, opt3,opt4, opt5, opt6, opt7) of the nucleic acid sequences as describedherein that encode the secretory signal peptide preferably having theamino acid sequence as defined by the SEQ ID NOs indicated in the thirdcolumn (“A”) or by the database entry indicated in the second column(“Accession No.”). Additional information regarding each of thesequences provided in Table 3 may also be derived from the sequencelisting, in particular from the details provided therein underidentifier <223>.

TABLE 3 List of suitable secretory signal peptides: NCBI Accession A B CD E F G H J Name No. Protein CDS wt CDS opt1 CDS opt2 CDS opt3 CDS opt4CDS opt5 CDS opt6 CDS opt7 HsHLA-A2(1-24) AAA59606 258 317 349 381 413445 477 509 541 HsPLAT(1-23) AAA61213 259 318 350 382 414 446 478 510542 HsPLAT(1-21) AAA61213 260 319 351 383 415 447 479 511 543HsPLAT(1-22) AAA61213 261 320 352 384 416 448 480 512 544 HsEPO(1-27)NP_000790 262 321 353 385 417 449 481 513 545 HsALB(1-18) NP_000468 263322 354 386 418 450 482 514 546 HsIgE(1-18) AAB59424 264 323 355 387 419451 483 515 547 HsCD5(1-24) NP_055022 265 324 356 388 420 452 484 516548 HsIL2(1-20) NP_000577 266 325 357 389 421 453 485 517 549HsCTRB2(1-18) NP_001020371 267 326 358 390 422 454 486 518 550HsIgG-HC(1-19) BAC87457 268 327 359 391 423 455 487 519 551HsIg-HC(1-19) AAA52897 269 328 360 392 424 456 488 520 552 HsIg-LC(1-19)AAA59018 270 329 361 393 425 457 489 521 553 GpLuc(1-17) AAG54095 271330 362 394 426 458 490 522 554 MmIgkappa(1-21) BAR42292 272 331 363 395427 459 491 523 555 NrChit1(1-26) ABF74624 273 332 364 396 428 460 492524 556 CILp1.1(1-21) AAS93426 274 333 365 397 429 461 493 525 557NgNep1(1-24) AB114914 275 334 366 398 430 462 494 526 558 HsAzu1(1-19)NP_001691 276 335 367 399 431 463 495 527 559 HsCD33(1-16) AAA51948 277336 368 400 432 464 496 528 560 VcCtxB(1-19) BAA06291 278 337 369 401433 465 497 529 561 HsCST4(1-20) NP_001890 279 338 370 402 434 466 498530 562 HsIns-iso1(1-24) AAA59172 280 339 371 403 435 467 499 531 563HsSPARC(1-17) CAA68724 281 340 372 404 436 468 500 532 564 H1N1-HA(1-17)ACQ45338 282 341 373 405 437 469 501 533 565 HsMHCII(1-25) CAA23783 310342 374 406 438 470 502 534 566 F(1-26) AAK50553 311 343 375 407 439 471503 535 567 F(1-26) AEZ01388 312 344 376 408 440 472 504 536 568 F(1-26)AAY43915 313 345 377 409 441 473 505 537 569 F(1-26) CBM41033 314 346378 410 442 474 506 538 570 F(1-26) NP_047111 315 347 379 411 443 475507 539 571 F(1-25) AEQ38114 316 348 380 412 444 476 508 540 572

Accordingly, in preferred embodiments, the at least one coding sequenceof the artificial nucleic acid of the invention additionally encodes atleast one further peptide or protein element selected from a secretorysignal peptide, wherein the secretory signal peptide comprises an aminoacid sequence being identical or at least 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 258-282, 310-316 (as provided inTable 3), or a fragment or variant of any of these sequences.

According to preferred embodiments, secretory signal peptides asprovided in Table 3 may be N-terminally fused to Hendra virus F_deISS orSoIG proteins as provided in Table 1B or Hendra virus F_deISS or SoIGproteins as provided in Table 2B to generate antigenic Hendra and Nipahvirus proteins optimized for secretion. Preferred embodiments of Hendraand Nipah virus proteins comprising heterologous N-terminal signalpeptides are provided in Table 1B and Table 2B.

According to preferred embodiments, the nucleic acid sequence accordingto the invention comprises at least one coding sequence encoding aheterologous secretory signal sequence as defined above and, inaddition, a Henipavirus antigenic peptide or protein being identical orat least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acidsequences according to SEQ ID NOs: 807-832, 1041-1066 or a fragment orvariant of any of these sequences.

Transmembrane Domains VLP Forming Domains Peptide Linker Self-CleavingPeptides Helper Peptides:

According to another embodiment, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one transmembrane domain element.Transmembrane elements or membrane spanning polypeptide elements arepresent in proteins that are integrated or anchored in plasma membranesof cells. Typical transmembrane elements are alpha-helical transmembraneelements. Such transmembrane elements are composed essentially of aminoacids with hydrophobic side chains, because the interior of a cellmembrane (lipid bilayer) is also hydrophobic. From the structuralperspective, transmembrane elements are commonly single hydrophobicalpha helices or beta barrel structures; whereas hydrophobic alphahelices are usually present in proteins that are present in membraneanchored proteins (e.g., seven transmembrane domain receptors),beta-barrel structures are often present in proteins that generate poresor channels. For target proteins, such as antigenic peptides or proteinsaccording to the present invention (derived from Henipavirus, Hendravirus, Nipah virus) it may be beneficial to introduce a transmembraneelement into the respective constructs. By addition of a transmembraneelement to the target peptide/protein it may be possible to furtherenhance the immune response, wherein the translated targetpeptide/protein, e.g. a viral antigen, anchors to a target membrane,e.g. the plasma membrane of a cell, thereby increasing immune responses.This effect is also referred to as antigen clustering. When used incombination with a polypeptide or protein of interest in the context ofthe present invention, such transmembrane element can be placedN-terminal or C-terminal to the Henipavirus and/or Hendra virus and/orNipah virus antigenic peptide or protein of interest. On nucleic acidlevel, the coding sequence for such transmembrane element is typicallyplaced in frame (i.e. in the same reading frame), 5′ or 3′ to the codingsequence of the polypeptide as defined herein. The transmembrane domainmay be selected from the transmembrane domain of Hemagglutinin (HA) ofInfluenza virus, Env of HIV-1. EIAV (equine infectious anaemia virus),MLV (murine leukaemia virus), mouse mammary tumor virus, G protein ofVSV (vesicular stomatitis virus), Rabies virus, or a transmembraneelement of a seven transmembrane domain receptor. Specific elementssuitable in the context of the present invention are provided in thesequence listing (SEQ ID NOs: 283-294). On nucleic acid level,particularly RNA level, any nucleotide sequence moiety can be employedthat encodes any transmembrane domain used in the present invention.Owing to the degenerated genetic code, in the case of most polypeptidesSEQ ID NOs: 283-294, more than one particular nucleic acid sequence isconceivable as encoding the respective polypeptide. While each and everysuch nucleic acid may generally be used in the context of the presentinvention, it is preferable that the nucleic acid sequence that encodesthe polypeptide sequence is selected such that its sequence is optimizedaccording to the general guidance provided in this specification.Alternatively, any polypeptide element may be selected which ischaracterized by at least 80% identity, at least 85% identity,preferably at least 90% identity, and more preferably at least 95%identity to any of the sequences SEQ ID NOs: 283-294. On nucleic acidlevel, any polynucleotide (e.g. RNA) moiety may be selected whichencodes such polypeptide element.

According to another embodiment, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one VLP forming domain. VLPs areself-assembled viral structural proteins (envelope proteins or capsidproteins) that structurally resemble viruses (without containing viralgenetic material). VLPs contain repetitive high density displays ofantigens which present conformational epitopes that can elicit strong Tcell and B cell immune responses. When used in combination with aHenipavirus and/or Hendra virus and/or Nipah virus antigenic peptide orprotein in the context of the present invention, such VLP formingelement can be placed N-terminal or C-terminal to the polypeptide ofinterest. On nucleic acid level, the coding sequence for such VLPforming element is typically placed in frame (i.e. in the same readingframe), 5′ or 3′ to the coding sequence of the polypeptide as definedherein. For nucleic acid (e.g. RNA) encoding a polypeptide or protein ofinterest, particularly antigenic polypeptides or proteins (Henipavirus,Hendra virus, Nipah virus), it may be beneficial to introduce a VLPforming element into the respective constructs. In addition to the“clustering” of epitopes, an improved secretion of the VLP particle mayalso increase the immunogenicity of the respective antigen. VLP formingelements fused to an antigen may generate virus like particlescontaining repetitive high density displays of antigens. VLP formingelements may be selected e.g. from any one of SEQ ID NOs: 295-296.Essentially, such VLP forming elements can be chosen from any viral orphage capsid or envelope protein. VLP forming elements may be used asadditional elements to promote or improve the particle formation of thetarget protein. Suitably, the polypeptide sequence of the VLP formingelement used in the present invention is selected from the followinglist of polypeptide sequences (SEQ ID NOs: 295-296). On nucleic acidlevel, particularly RNA level, any nucleotide sequence moiety can beemployed that encodes any of VLP forming element used in the presentinvention. Owing to the degenerated genetic code, in the case of mostpolypeptides SEQ ID NOs: 295-296, more than one particular nucleic acidsequence is conceivable as encoding the respective polypeptide of thebelow list. While each and every such nucleic acid may generally be usedin the context of the present invention, it is preferable that thenucleic acid sequence that encodes the polypeptide sequence is selectedsuch that its sequence is codon-optimized according to the generalguidance provided in this specification. Alternatively, any polypeptideelement may be selected which is characterized by at least 80% identity,at least 85% identity, preferably at least 90% identity, and morepreferably at least 95% identity to any of the sequences SEQ ID NOs:295-296. On nucleic acid level, any polynucleotide (e.g. RNA) moiety maybe selected which encodes such polypeptide element.

According to another embodiment, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one peptide linker element. In proteinconstructs composed of several elements (e.g., Henipavirus antigenicpeptide or protein fused to a transmembrane domain), the proteinelements may be separated by peptide linker elements. Such elements maybe beneficial because they allow for a proper folding of the individualelements and thereby the proper functionality of each element.Alternatively, the term “spacer” or “peptide spacer” is used herein.When used in the context of the present invention, such linkers orspacers are particularly useful when encoded by a nucleic acid encodingat least two functional protein elements, such as at least onepolypeptide or protein of interest (Nipah virus and/or Hendra virusantigens) and at least one further protein or polypeptide element (e.g.,VLP forming domain, transmembrane domain). In that case, the linker istypically located on the polypeptide chain in between the polypeptide ofinterest and the at least one further protein element. On nucleic acidlevel, the coding sequence for such linker is typically placed in thereading frame, 5′ or 3′ to the coding sequence for the polypeptide orprotein of interest, or placed between coding regions for individualpolypeptide domains of a given protein of interest. Peptide linkers arepreferably composed of small, non-polar (e.g. Gly) or polar (e.g. Ser orThr) amino acids. The small size of these amino acids providesflexibility, and allows for mobility of the connecting functionaldomains. The incorporation of Ser or Thr can maintain the stability ofthe linker in aqueous solutions by forming hydrogen bonds with the watermolecules, and therefore reduces an interaction between the linker andthe protein moieties. Rigid linkers generally maintain the distancebetween the protein domains and they may be based on helical structuresand/or they have a sequence that is rich in proline. Cleavable linkers(also termed “cleavage linkers”) allow for in vivo separation of theprotein domains. The mechanism of cleavage may be based e.g. onreduction of disulfide bonds within the linker sequence or proteolyticcleavage. The cleavage may be mediated by an enzyme (enzymaticcleavage), e.g. the cleavage linker may provide a protease sensitivesequence (e.g., furin cleavage). A typical sequence of a flexible linkeris composed of repeats of the amino acids Glycine (G) and Serine (S).For instance, the linker may have the following sequence: GS, GSG, SGG,SG, GGS, SGS, GSS, SSG. In some embodiments, the same sequence isrepeated multiple times (e.g. two, three, four, five or six times) tocreate a longer linker. In other embodiments, a single amino acidresidue such as S or G can be used as a linker. Linkers or spacers maybe used as additional elements to promote or improve the secretion ofthe target protein (Henipavirus and/or Hendra virus and/or Nipah virusantigenic peptides or proteins). Suitably, the polypeptide sequence ofthe linker or spacer used in the present invention is selected from thefollowing list of polypeptide sequences (SEQ ID NOs: 297-299). Onnucleic acid level, particularly RNA level, any nucleotide sequencemoiety can be employed that encodes any of linker or spacer used in thepresent invention. Owing to the degenerated genetic code, in the case ofmost polypeptides of SEQ ID NOs: 297-299, more than one particularnucleic acid sequence is conceivable as encoding the respectivepolypeptide list. While each and every such nucleic acid may generallybe used in the context of the present invention, it is preferable thatthe nucleic acid sequence that encodes the polypeptide sequence isselected such that its sequence is optimized according to the generalguidance provided in this specification. Alternatively, any polypeptideelement may be selected which is characterized by at least 80% identity,at least 85% identity, preferably at least 90% identity, and morepreferably at least 95% identity to any of the sequences SEQ ID NOs:297-299. On nucleic acid level, any polynucleotide (e.g. RNA) moiety maybe selected which encodes such polypeptide element.

According to another embodiment, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one self-cleaving peptide. Viralself-cleaving peptides (2A peptides) allow the expression of multipleproteins from a single open reading frame. The terms 2A peptide and 2Aelement are used interchangeably herein. The mechanism by the 2Asequence for generating two proteins from one transcript is by ribosomeskipping—a normal peptide bond is impaired at 2A, resulting in twodiscontinuous protein fragments from one translation event. When used inthe context of the present invention, such 2A peptides are particularlyuseful when encoded by a nucleic acid encoding at least two functionalprotein elements (e.g. Henipavirus and/or Hendra virus and/or Nipahvirus antigenic peptides or proteins). In general, a 2A element isuseful when the nucleic acid molecule encodes at least one polypeptideor protein of interest and at least one further protein element. In apreferred embodiment, a 2A element is present when the polynucleotide ofthe invention encodes two proteins or polypeptides of interest, e.g. twoantigens. The coding sequence for such 2A peptide is typically locatedin between the coding sequence of the polypeptide of interest and thecoding sequence of the least one further protein element (which may alsobe a polypeptide of interest), so that cleavage of the 2A peptide leadsto two separate polypeptide molecules, at least one of them being apolypeptide or protein of interest. For example, for expressing targetproteins (Henipavirus and/or Hendra virus and/or Nipah virus antigenicpeptides or proteins) that are composed of several polypeptide chains,such as antibodies, it may be beneficial to provide coding informationfor both polypeptide chains on a single nucleic acid molecule, separatedby a nucleic acid sequence encoding a 2A peptide. 2A peptides may alsobe beneficial when cleavage of the protein of interest from anotherencoded polypeptide element is desired. 2A peptides may be derived fromfoot-and-mouth diseases virus, from equine rhinitis A virus, Thoseaasigna virus, Porcine teschovirus-1. Suitably, the polypeptide sequenceof the 2A peptide used in the present invention may be selected from thefollowing list of polypeptide sequences (SEQ ID NOs: 300-303). Onnucleic acid level, particularly RNA level, any nucleotide sequencemoiety can be employed that encodes any of 2A peptide used in thepresent invention. Owing to the degenerated genetic code, in the case ofmost polypeptides (SEQ ID NOs: 300-303), more than one particularnucleic acid sequence is conceivable as encoding the respectivepolypeptide. While each and every such nucleic acid may generally beused in the context of the present invention, it is preferable that thenucleic acid sequence that encodes the polypeptide sequence is selectedsuch that its sequence is optimized according to the general guidanceprovided in this specification. Alternatively, any polypeptide elementmay be selected which is characterized by at least 80% identity, atleast 85% identity, preferably at least 90% identity, and morepreferably at least 95% identity to any of the sequences SEQ ID NOs:300-303. On nucleic acid level, any polynucleotide (e.g. RNA) moiety maybe selected which encodes such polypeptide element.

According to another embodiment, the artificial nucleic acid sequence,particularly the RNA sequence according to the invention mayadditionally encode at least one helper peptide. In essence, helperpeptides binds to class II MHC molecules as a nonspecific vaccine helperepitope (adjuvant) and induces an increased (and long term) immuneresponse by increasing the helper T-cell response. In an embodiment,such a helper peptide may be N-terminally and/or C-terminally fused tothe antigenic peptide or protein derived from Henipavirus, Nipah virusor Hendra virus. In an embodiment, the helper peptide is derived fromtetanus toxin, according to SEQ ID NO: 257. On nucleic acid level,particularly RNA level, any nucleotide sequence moiety can be employedthat encodes any helper peptide used in the present invention. Owing tothe degenerated genetic code, in the case of most polypeptides (SEQ IDNO: 257), more than one particular nucleic acid sequence is conceivableas encoding the respective polypeptide. While each and every suchnucleic acid may generally be used in the context of the presentinvention, it is preferable that the nucleic acid sequence that encodesthe polypeptide sequence is selected such that its sequence is optimizedaccording to the general guidance provided in this specification.Alternatively, any polypeptide element may be selected which ischaracterized by at least 80% identity, at least 85% identity,preferably at least 90% identity, and more preferably at least 95%identity to any of the sequences SEQ ID NO: 257. On nucleic acid level,any polynucleotide (e.g. RNA) moiety may be selected which encodes suchpolypeptide element.

Henipavirus Nucleic Acids:

In the context of the invention, the coding sequence encoding the atleast one Henipavirus antigenic peptide or protein or fragment, variantor derivative thereof, may be selected from any nucleic acid sequencecomprising a coding sequence encoding Henipavirus RNA-directed RNApolymerase (L), Henipavirus fusion protein (F), Henipavirusnon-structural protein (V), Henipavirus glycoprotein (G), Henipavirusnucleoprotein (N), Henipavirus matrix protein (M), Henipavirusphosphoprotein (P), Henipavirus protein C, and Henipavirus protein W. Inthe context of the invention, said artificial nucleic acid sequences maybe derived from any Henipavirus strain, species, serotype, subtypefragment or variant thereof (e.g. as provided above in the section“Henipavirus”).

The artificial nucleic acid of the invention may comprise or consist ofat least one coding sequence encoding at least one Henipavirus antigenicpeptide or protein as defined herein, preferably encoding any one of SEQID NOs: 1-26, 573-598, 807-832, 1041-1066, 1513-1515 or fragments ofvariants thereof. It has to be understood that, on nucleic acid level,any nucleic acid sequence (e.g. DNA sequence, RNA sequence) whichencodes an amino acid sequences being identical to SEQ ID NOs: 1-26,573-598, 807-832, 1041-1066, 1513-1515 or fragments or variants thereof,or any nucleic acid sequence (e.g. DNA sequence, RNA sequence) whichencodes amino acid sequences being at least 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to any one of SEQ ID NOs: 1-26, 573-598, 807-832, 1041-1066,1513-1515 or fragments or variants thereof, may be selected and mayaccordingly be understood as suitable coding sequence and may thereforebe comprised in the artificial nucleic acid of the invention.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Henipavirus antigenic peptide or protein as described herein.Preferably, the inventive artificial nucleic acid comprises or consistsof a coding sequence according to any one of SEQ ID NOs: 27-234,599-806, 833-1040, 1067-1274, 1275-1508, 1516-1539, 1540-1548 or ahomolog, fragment or variant of any of these sequences.

The artificial nucleic acid according to any one of the precedingclaims, wherein the at least one coding sequence comprises at least oneof the RNA sequences being identical or at least 50%, 60%, 70%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identical to SEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274,1275-1508, 1516-1539, 1540-1548 or at least one of the RNA sequenceswhich are capable of hybridizing with a complement sequence derived fromSEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274, 1275-1508, 1516-1539,1540-1548 or a fragment or variant or orthologue or paralogue of any ofthese.

It is further preferred that the nucleic acid sequence according to theinvention comprises at least one coding sequence encoding a heterologoussecretory signal sequence comprising a nucleic acid sequence selectedfrom sequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleic acid sequences according to SEQ ID NOs 317-572or a fragment or variant thereof and, in addition, at least one codingsequence encoding a Henipavirus antigenic peptide or protein comprisinga nucleic acid sequence selected from sequences being identical or atleast 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequencesaccording to SEQ ID NOs: 599-806 or a fragments or variants any of thesesequences.

In this context, it is preferred that the nucleic acid sequenceaccording to the invention comprises at least one coding sequenceencoding a heterologous secretory signal sequence and, in addition, atleast one coding sequence encoding a Henipavirus antigenic peptide orprotein comprising a nucleic acid sequence selected from sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleicacid sequences according to SEQ ID NOs: 833-1040, 1067-1274, 1516-1539or a fragments or variants any of these sequences.

Hendra Virus Nucleic Acids:

In the context of the invention, the coding sequence encoding the atleast one Hendra virus antigenic peptide or protein or fragment, variantor derivative thereof, may be selected from any nucleic acid sequencecomprising a coding sequence encoding Hendra virus RNA-directed RNApolymerase (L), Hendra virus fusion protein (F), Hendra virusnon-structural protein (V), Hendra virus glycoprotein (G), Hendra virusnucleoprotein (N), Hendra virus matrix protein (M), Hendra virusphosphoprotein (P), Hendra virus protein C, and Hendra virus protein W.In the context of the invention, said artificial nucleic acid sequencesmay be derived from any Hendra virus strain, species, serotype, subtypefragment or variant thereof.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Hendra virus antigenic peptide or protein as described herein.Preferably, the inventive artificial nucleic acid comprises or consistsof a coding sequence according to any one of SEQ ID NOs: 34-37, 45-52,60-63, 71-78, 86-89, 97-104, 112-115, 123-130, 138-141, 149-156,164-167, 175-182, 190-193, 201-208, 216-219, 227-234, 606-609, 632-635,658-661, 684-687, 710-713, 736-739, 762-765, 788-791, 617-624, 643-650,669-676, 695-702, 721-728, 747-754, 773-780, 799-806, 840-843, 866-869,892-895, 918-921, 944-947, 970¬-973, 996-999, 1022-1025, 851-858,877-884, 903-910, 929-936, 955-962, 981-988, 1007-1014, 1033-1040,1074-1077, 1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207,1230-1233, 1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170,1189-1196, 1215-1222, 1241-1248, 1267-1274, 1282-1285, 1293-1300,1308-1311, 1319-1326, 1334-1337, 1345-1352, 1360-1363, 1371-1378,1386-1389, 1397-1404, 1412-1415, 1423-1430, 1438-1441, 1464-1467,1490-1493, 1449-1456, 1475-1482, 1501-1508 or a homolog, fragment orvariant of any of these sequences.

Preferably, the nucleic acid sequence according to the inventioncomprises at least one coding sequence encoding Hendra virus antigenicpeptide or protein comprising a nucleic acid sequence selected fromsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the nucleic acid sequences according to SEQ ID NOs: 34-37, 45-52,60-63, 71-78, 86-89, 97-104, 112-115, 123-130, 138-141, 149-156,164-167, 175-182, 190-193, 201-208, 216-219, 227-234, 606-609, 632-635,658-661, 684-687, 710-713, 736-739, 762-765, 788-791, 617-624, 643-650,669-676, 695-702, 721-728, 747-754, 773-780, 799-806, 840-843, 866-869,892-895, 918-921, 944-947, 970¬-973, 996-999, 1022-1025, 851-858,877-884, 903-910, 929-936, 955-962, 981-988, 1007-1014, 1033-1040,1074-1077, 1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207,1230-1233, 1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170,1189-1196, 1215-1222, 1241-1248, 1267-1274, 1282-1285, 1293-1300,1308-1311, 1319-1326, 1334-1337, 1345-1352, 1360-1363, 1371-1378,1386-1389, 1397-1404, 1412-1415, 1423-1430, 1438-1441, 1464-1467,1490-1493, 1449-1456, 1475-1482, 1501-1508 or a homolog, fragment orvariant of any of these sequences, or at least one of the nucleic acidsequences which are capable of hybridizing with a complement sequencederived from SEQ ID NOs: 34-37, 45-52, 60-63, 71-78, 86-89, 97-104,112-115, 123-130, 138-141, 149-156, 164-167, 175-182, 190-193, 201-208,216-219, 227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739,762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728, 747-754,773-780, 799-806, 840-843, 866-869, 892-895, 918-921, 944-947, 970¬-973,996-999, 1022-1025, 851-858, 877-884, 903-910, 929-936, 955-962,981-988, 1007-1014, 1033-1040, 1074-1077, 1100-1103, 1126-1129,1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259, 1085-1092,1111-1118, 1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248,1267-1274, 1282-1285, 1293-1300, 1308-1311, 1319-1326, 1334-1337,1345-1352, 1360-1363, 1371-1378, 1386-1389, 1397-1404, 1412-1415,1423-1430, 1438-1441, 1464-1467, 1490-1493, 1449-1456, 1475-1482,1501-1508 or a fragment or variant or orthologue or paralogue of any ofthese.

In a preferred embodiment, the present invention thus providesartificial nucleic acid sequences comprising at least one codingsequence, wherein the coding sequence encoding Hendra virus fusionprotein (F) comprises or consists of any one of the nucleic acidsequences defined in Table 1 and Table 1B, a homolog, fragment orvariant of any one of these sequences.

In particularly preferred embodiments the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Hendravirus fusion protein (F) according to SEQ ID NOs: 34-37, 60-63, 86-89,112-115, 138-141, 164-167, 190-193, 216-219, 606-609, 632-635, 658-661,684-687, 710-713, 736-739, 762-765, 788-791, 840-843, 866-869, 892-895,918-921, 944-947, 970-973, 996-999, 1022-1025, 1074-1077, 1100-1103,1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259, or ahomolog, fragment or variant of any of these sequences.

Preferably, the nucleic acid sequence according to the inventioncomprises at least one coding sequence encoding Hendra virus fusionprotein (F) comprising a nucleic acid sequence selected from sequencesbeing identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thenucleic acid sequences according to SEQ ID NOs: 34-37, 60-63, 86-89,112-115, 138-141, 164-167, 190-193, 216-219, 606-609, 632-635, 658-661,684-687, 710-713, 736-739, 762-765, 788-791, 840-843, 866-869, 892-895,918-921, 944-947, 970-973, 996-999, 1022-1025, 1074-1077, 1100-1103,1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259 and asdisclosed in Table 1 and Table 1B.

In a preferred embodiment, the present invention thus providesartificial nucleic acid sequences comprising at least one codingsequence, wherein the coding sequence encoding Hendra virus glycoprotein(G) comprises or consists of any one of the nucleic acid sequencesdefined in Table 1 and Table 1B, a homolog, fragment or variant of anyone of these sequences.

In particularly preferred embodiments the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Hendravirus glycoprotein (G) according to SEQ ID NOs: 45-52, 71-78, 97-104,123-130, 149-156, 175-182, 201-208, 227-234, 617-624, 643-650, 669-676,695-702, 721-728, 747-754, 773-780, 799-806, 851-858, 877-884, 903-910,929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1085-1092, 1111-1118,1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248, 1267-1274, or ahomolog, fragment or variant of any of these sequences.

Preferably, the nucleic acid sequence according to the inventioncomprises at least one coding sequence encoding Hendra virusglycoprotein (G) comprising a nucleic acid sequence selected fromsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the nucleic acid sequences according to SEQ ID NOs: 45-52, 71-78,97-104, 123-130, 149-156, 175-182, 201-208, 227-234, 617-624, 643-650,669-676, 695-702, 721-728, 747-754, 773-780, 799-806, 851-858, 877-884,903-910, 929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1085-1092,1111-1118, 1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248,1267-1274, and as disclosed in Table 1 and Table 1B.

It is further preferred that the nucleic acid sequence according to theinvention comprises at least one coding sequence encoding a heterologoussecretory signal sequence comprising a nucleic acid sequence selectedfrom sequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleic acid sequences according to SEQ ID NOs: 317-572or a fragment or variant thereof and, in addition, at least one codingsequence encoding a Hendra virus antigenic peptide or protein comprisinga nucleic acid sequence selected from sequences being identical or atleast 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequencesaccording to SEQ ID NOs: 606-609, 632-635, 658-661, 684-687, 710-713,736-739, 762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728,747-754, 773-780, 799-806 or a homolog, fragment or variant of any ofthese sequences.

In this context, it is preferred that the nucleic acid sequenceaccording to the invention comprises at least one coding sequenceencoding a heterologous secretory signal sequence and, in addition, atleast one coding sequence encoding a Hendra virus antigenic peptide orprotein comprising a nucleic acid sequence selected from sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleicacid sequences according to SEQ ID NOs: 840-843, 866-869, 892-895,918-921, 944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884,903-910, 929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077,1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233,1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196,1215-1222, 1241-1248, 1267-1274 or a homolog, fragment or variant of anyof these sequences.

In another embodiment the nucleic acid sequence comprises or consists ofat least one coding sequence encoding Hendra virus RNA-directed RNApolymerase (L), or a fragment or variant thereof. In another embodimentthe nucleic acid sequence comprises or consists of at least one codingsequence encoding Hendra virus non-structural protein (V), or a fragmentor variant thereof. In another embodiment the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Hendravirus nucleoprotein (N), or a fragment or variant thereof. In anotherembodiment the nucleic acid sequence comprises or consists of at leastone coding sequence encoding Hendra virus matrix protein (M), or afragment or variant thereof. In another embodiment the nucleic acidsequence comprises or consists of at least one coding sequence encodingHendra virus phosphoprotein (P), or a fragment or variant thereof. Inanother embodiment the nucleic acid sequence comprises or consists of atleast one coding sequence encoding Hendra virus protein C, or a fragmentor variant thereof. In another embodiment the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Hendravirus protein W, or a fragment or variant thereof.

The inventive artificial nucleic acid encoding Hendra virus antigenicpeptide or protein, preferably the at least one coding sequence of theartificial nucleic acid according to the invention, may comprise orconsist of a variant of a nucleic acid sequence as defined herein,preferably of a nucleic acid sequence encoding a protein or a fragmentthereof as defined herein. The expression “variant of a nucleic acidsequence” as used herein in the context of a nucleic acid sequenceencoding a protein or a fragment thereof, typically refers to a nucleicacid sequence, which differs by at least one nucleic acid residue fromthe respective naturally occurring nucleic acid sequence encoding aprotein or a fragment thereof. More preferably, the expression “variantof a nucleic acid sequence” refers to a nucleic acid sequence having asequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, preferably of at least 70%, more preferably of at least80%, even more preferably at least 85%, even more preferably of at least90% and most preferably of at least 95% or even 97%, with a nucleic acidsequence, from which it is derived.

Nipah Virus Nucleic Acids:

In the context of the invention, the coding sequence encoding the atleast one Nipah virus antigenic peptide or protein or fragment, variantor derivative thereof, may be selected from any nucleic acid sequencecomprising a coding sequence encoding Nipah virus RNA-directed RNApolymerase (L), Nipah virus fusion protein (F), Nipah virusnon-structural protein (V), Nipah virus glycoprotein (G), Nipah virusnucleoprotein (N), Nipah virus matrix protein (M), Nipah virusphosphoprotein (P), Nipah virus protein C, and Nipah virus protein W. Inthe context of the invention, said artificial nucleic acid sequences maybe derived from any Nipah virus strain, species, serotype, subtypefragment or variant thereof.

According to a preferred embodiment, the inventive artificial nucleicacid comprises or consists of at least one coding sequence encoding atleast one Nipah virus antigenic peptide or protein as described herein.Preferably, the inventive artificial nucleic acid comprises or consistsof a coding sequence according to any one of SEQ ID NOs: 27-33, 38-44,53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137, 142-148, 157-163,168-174, 183-189, 194-200, 209-215, 220-226, 599-605, 625-631, 651-657,677-683, 703-709, 729-735, 755-761, 781-787, 610-616, 636-642, 662-668,688-694, 714-720, 740-746, 766-772, 792-798, 833-839, 859-865, 885-891,911-917, 937-943, 963-969, 989-995, 1015-1021, 844-850, 870-876,896-902, 922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1067-1073,1093-1099, 1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229,1249-1255, 1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188,1208-1214, 1234-1240, 1260-1266, 1275-1281, 1286-1292, 1301-1307,1312-1318, 1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385,1390-1396, 1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489,1442-1448, 1468-1474, 1494-1500, 1516-1539, 1540-1548 or a homolog,fragment or variant of any of these sequences.

Preferably, the nucleic acid sequence according to the inventioncomprises at least one coding sequence encoding Nipah virus antigenicpeptide or protein comprising a nucleic acid sequence selected fromsequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identicalto the nucleic acid sequences according to SEQ ID NOs: 27-33, 38-44,53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137, 142-148, 157-163,168-174, 183-189, 194-200, 209-215, 220-226, 599-605, 625-631, 651-657,677-683, 703-709, 729-735, 755-761, 781-787, 610-616, 636-642, 662-668,688-694, 714-720, 740-746, 766-772, 792-798, 833-839, 859-865, 885-891,911-917, 937-943, 963-969, 989-995, 1015-1021, 844-850, 870-876,896-902, 922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1067-1073,1093-1099, 1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229,1249-1255, 1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188,1208-1214, 1234-1240, 1260-1266, 1275-1281, 1286-1292, 1301-1307,1312-1318, 1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385,1390-1396, 1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489,1442-1448, 1468-1474, 1494-1500, 1516-1539, 1540-1548 or a homolog,fragment or variant of any of these sequences, or at least one of thenucleic acid sequences which are capable of hybridizing with acomplement sequence derived from SEQ ID NOs: 27-33, 38-44, 53-59, 64-70,79-85, 90-96, 105-111, 116-122, 131-137, 142-148, 157-163, 168-174,183-189, 194-200, 209-215, 220-226, 599-605, 625-631, 651-657, 677-683,703-709, 729-735, 755-761, 781-787, 610-616, 636-642, 662-668, 688-694,714-720, 740-746, 766-772, 792-798, 833-839, 859-865, 885-891, 911-917,937-943, 963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902,922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099,1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214,1234-1240, 1260-1266, 1275-1281, 1286-1292, 1301-1307, 1312-1318,1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385, 1390-1396,1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489, 1442-1448,1468-1474, 1494-1500, 1516-1539, 1540-1548 or a fragment or variant ororthologue or paralogue of any of these.

In a preferred embodiment, the present invention provides artificialnucleic acid sequences comprising at least one coding sequence, whereinthe coding sequence encoding Nipah virus fusion protein (F) comprises orconsists of any one of the nucleic acid sequences defined in Table 2 andTable 2B, a homolog, fragment or variant of any one of these sequences.

In particularly preferred embodiments the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Nipahvirus fusion protein (F) according to SEQ ID NOs: 27-33, 53-59, 79-85,105-111, 131-137, 157-163, 183-189, 209-215, 599-605, 625-631, 651-657,677-683, 703-709, 729-735, 755-761, 781-787, 833-839, 859-865, 885-891,911-917, 937-943, 963-969, 989-995, 1015-1021, 1067-1073, 1093-1099,1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,1516-1539 or a homolog, fragment or variant of any of these sequences.

Preferably, the nucleic acid sequence according to the inventioncomprises at least one coding sequence encoding Nipah virus fusionprotein (F) comprising a nucleic acid sequence selected from sequencesbeing identical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to thenucleic acid sequences according to SEQ ID NOs: 27-33, 53-59, 79-85,105-111, 131-137, 157-163, 183-189, 209-215, 599-605, 625-631, 651-657,677-683, 703-709, 729-735, 755-761, 781-787, 833-839, 859-865, 885-891,911-917, 937-943, 963-969, 989-995, 1015-1021, 1067-1073, 1093-1099,1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,1516-1539 and as disclosed in Table 2 and Table 2B.

In a preferred embodiment, the present invention provides artificialnucleic acid sequences comprising at least one coding sequence, whereinthe coding sequence encoding Nipah virus glycoprotein (G) comprises orconsists of any one of the nucleic acid sequences defined in Table 2 andTable 2B, a homolog, fragment or variant of any one of these sequences.

In particularly preferred embodiments the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Nipahvirus glycoprotein (G) according to SEQ ID NOs: 38-44, 64-70, 90-96,116-122, 142-148, 168-174, 194-200, 220-226, 610-616, 636-642, 662-668,688-694, 714-720, 740-746, 766-772, 792-798, 844-850, 870-876, 896-902,922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1078-1084, 1104-1110,1130-1136, 1156-1162, 1182-1188, 1208-1214, 1234-1240, 1260-1266, or ahomolog, fragment or variant of any of these sequences.

Preferably, the nucleic acid sequence according to the inventioncomprises at least one coding sequence encoding Nipah virus glycoprotein(G) comprising a nucleic acid sequence selected from sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleicacid sequences according to SEQ ID NOs: 38-44, 64-70, 90-96, 116-122,142-148, 168-174, 194-200, 220-226, 610-616, 636-642, 662-668, 688-694,714-720, 740-746, 766-772, 792-798, 844-850, 870-876, 896-902, 922-928,948-954, 974-980, 1000-1006, 1026-1032, 1078-1084, 1104-1110, 1130-1136,1156-1162, 1182-1188, 1208-1214, 1234-1240, 1260-1266, and as disclosedin Table 2 and Table 2B.

It is further preferred that the nucleic acid sequence according to theinvention comprises at least one coding sequence encoding a heterologoussecretory signal sequence comprising a nucleic acid sequence selectedfrom sequences being identical or at least 50%, 60%, 70%, 80%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to the nucleic acid sequences according to SEQ ID NOs: 317-572or a fragment or variant thereof and, in addition, at least one codingsequence encoding a Nipah virus antigenic peptide or protein comprisinga nucleic acid sequence selected from sequences being identical or atleast 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequencesaccording to SEQ ID NOs: 599-605, 625-631, 651-657, 677-683, 703-709,729-735, 755-761, 781-787, 610-616, 636-642, 662-668, 688-694, 714-720,740-746, 766-772, 792-798 or a homolog, fragment or variant of any ofthese sequences.

In this context, it is preferred that the nucleic acid sequenceaccording to the invention comprises at least one coding sequenceencoding a heterologous secretory signal sequence and, in addition, atleast one coding sequence encoding a Nipah virus antigenic peptide orprotein comprising a nucleic acid sequence selected from sequences beingidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleicacid sequences according to SEQ ID NOs: 833-839, 859-865, 885-891,911-917, 937-943, 963-969, 989-995, 1015-1021, 844-850, 870-876,896-902, 922-928, 948-954, 974-980, 1000-1006, 1026-1032, 1067-1073,1093-1099, 1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229,1249-1255, 1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188,1208-1214, 1234-1240, 1260-1266, 1516-1539 or a homolog, fragment orvariant of any of these sequences.

In another embodiment the nucleic acid sequence comprises or consists ofat least one coding sequence encoding Nipah virus RNA-directed RNApolymerase (L), or a fragment or variant thereof. In another embodimentthe nucleic acid sequence comprises or consists of at least one codingsequence encoding Nipah virus non-structural protein (V), or a fragmentor variant thereof. In another embodiment the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Nipahvirus nucleoprotein (N), or a fragment or variant thereof. In anotherembodiment the nucleic acid sequence comprises or consists of at leastone coding sequence encoding Nipah virus matrix protein (M), or afragment or variant thereof. In another embodiment the nucleic acidsequence comprises or consists of at least one coding sequence encodingNipah virus phosphoprotein (P), or a fragment or variant thereof. Inanother embodiment the nucleic acid sequence comprises or consists of atleast one coding sequence encoding Nipah virus protein C, or a fragmentor variant thereof. In another embodiment the nucleic acid sequencecomprises or consists of at least one coding sequence encoding Nipahvirus protein W, or a fragment or variant thereof.

The inventive artificial nucleic acid encoding Nipah virus antigenicpeptide or protein, preferably the at least one coding sequence of theartificial nucleic acid according to the invention, may comprise orconsist of a variant of a nucleic acid sequence as defined herein,preferably of a nucleic acid sequence encoding a protein or a fragmentthereof as defined herein. The expression “variant of a nucleic acidsequence” as used herein in the context of a nucleic acid sequenceencoding a protein or a fragment thereof, typically refers to a nucleicacid sequence, which differs by at least one nucleic acid residue fromthe respective naturally occurring nucleic acid sequence encoding aprotein or a fragment thereof. More preferably, the expression “variantof a nucleic acid sequence” refers to a nucleic acid sequence having asequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, preferably of at least 70%, more preferably of at least80%, even more preferably at least 85%, even more preferably of at least90% and most preferably of at least 95% or even 97%, with a nucleic acidsequence, from which it is derived.

In specific embodiments, the at least one coding sequence comprises atleast one of the DNA sequences being identical or at least 50%, 60%,70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to any one of the sequences SEQ ID NOs:27-234, 599-806, 833-1040, 1067-1274, 1275-1508, 1516-1539, 1540-1548wherein the indicated uridine nucleotides are substituted with thymidinenucleotides, or a fragment or variant or orthologue or paralogue of anyof these.

Mono-, Bi- and Multicistronic and Multi-Antigen Nucleic Acids:

According to certain embodiments of the present invention, theartificial nucleic acid is mono-, bi-, or multicistronic, preferably asdefined herein.

In specific embodiments the artificial nucleic acid of the invention ismonocistronic, wherein the (one) coding sequence encodes at least twodifferent Hendra virus and/or Nipah virus antigenic peptides orproteins, or a fragment or variant thereof.

The coding sequences in a bi- or multicistronic nucleic acid moleculepreferably encode distinct Henipavirus antigenic proteins or peptides,Hendra virus antigenic proteins or peptides, Nipah virus antigenicproteins or peptides as defined herein or a fragment or variant thereof.Preferably, the coding sequences encoding two or more antigenic proteinsor peptides may be separated in the bi- or multicistronic nucleic acidby at least one IRES (internal ribosomal entry site) sequence, asdefined below.

In specific embodiments the artificial nucleic acid of the invention isbi- or multicistronic and comprises at least two coding sequences,wherein the at least two coding sequences encode at least two differentHendra virus and/or Nipah virus antigenic peptides or proteins, or afragment or variant of any of these.

Thus, the term “encoding two or more antigenic peptides or proteins” or“encode at least two different Hendra virus and/or Nipah virus antigenicpeptides or proteins” may mean, without being limited thereto, that thebi- or even multicistronic nucleic acid, may encode e.g. at least two,three, four, five, six or more (preferably different) Henipavirusantigenic peptides or proteins and/or Hendra virus antigenic proteins orpeptides and/or Nipah virus antigenic proteins or peptides derived fromdifferent viruses or their fragments or variants within the definitionsprovided herein. More preferably, without being limited thereto, the bi-or even multicistronic nucleic acid, may encode, for example, at leasttwo, three, four, five, six or more (preferably different) Henipavirusantigenic peptides or proteins and/or Hendra virus antigenic proteins orpeptides and/or Nipah virus antigenic proteins or peptides as definedherein or their fragments or variants as defined herein. In thiscontext, a so-called IRES (internal ribosomal entry site) sequence asdefined above can function as a sole ribosome binding site, but it canalso serve to provide a bi- or even multicistronic nucleic acid asdefined above, which encodes several Henipavirus antigenic peptides orproteins and/or Hendra virus antigenic proteins or peptides and/or Nipahvirus antigenic proteins or peptides which are to be translated by theribosomes independently of one another. Examples of IRES sequences,which can be used according to the invention, are those frompicornaviruses (e.g. FMDV), pestiviruses (CFFV), polioviruses (PV),encephalomyocarditis viruses (ECMV), foot and mouth disease viruses(FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV),mouse leukoma virus (MLV), simian immunodeficiency viruses (Sly) orcricket paralysis viruses (CrPV).

Particular suitable IRES sequences that may be used in the context ofthe invention may be IRES sequences derived from EMCV (SEQ ID NO: 304)and IRES sequences derived from FMDV (SEQ ID NO: 305).

According to a further embodiment the at least one coding sequence ofthe nucleic acid sequence according to the invention may encode at leasttwo, three, four, five, six, seven, eight and more Henipavirus antigenicpeptides or proteins and/or Hendra virus antigenic proteins or peptidesand/or Nipah virus antigenic proteins or peptides (or fragments andderivatives thereof) as defined herein linked with or without an aminoacid linker sequence, wherein said linker sequence can comprise rigidlinkers, flexible linkers, cleavable linkers (e.g., self-cleavingpeptides) or a combination thereof (see paragraph “Peptide linkerelements” of the present invention). Therein, the Henipavirus antigenicpeptides or proteins and/or Hendra virus antigenic proteins or peptidesand/or Nipah virus antigenic proteins or peptides may be identical ordifferent or a combination thereof. Particular Henipavirus antigenicpeptides or proteins and/or Hendra virus antigenic proteins or peptidesand/or Nipah virus antigenic proteins or peptides combinations can beencoded by said nucleic acid encoding at least two Henipavirus antigenicpeptides or proteins and/or Hendra virus antigenic proteins or peptidesand/or Nipah virus antigenic proteins or peptides as explained herein(also herein referred to as “multi-antigen-constructs/nucleic acid”).

It has to be noted, that in the context of the invention, certaincombinations of coding sequences (e.g., comprising at least twodifferent Henipavirus antigenic peptides or proteins and/or Hendra virusantigenic proteins or peptides and/or Nipah virus antigenic proteins orpeptides and/or comprising at least two antigenic peptides or proteinsderived from a genetically different Henipavirus, Hendra virus, Nipahvirus) may be generated by any combination of moni- bi- andmulticistronic nucleic acids and/or multi-antigen-constructs/nucleicacid to obtain a poly- or even multivalent nucleic acid mixture.

Preferably, the at least one coding sequence of the nucleic acidsequence according to the invention comprises at least two, three, four,five, six, seven, eight or more nucleic acid sequences identical to orhaving a sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99%, preferably of at least 70%, more preferably of atleast 80%, even more preferably at least 85%, even more preferably of atleast 90% and most preferably of at least 95% or even 97%, with any oneof the nucleic acid sequences disclosed according to SEQ ID NOs: 27-234,599-806, 833-1040, 1067-1274, 1275-1508, 1516-1539, 1540-1548 or afragment or variant of any one of said nucleic acid sequences.

Preferably, the nucleic acid sequence comprising at least one codingsequence as defined herein typically comprises a length of about 50 toabout 20000, or 100 to about 20000 nucleotides, preferably of about 250to about 20000 nucleotides, more preferably of about 500 to about 10000,even more preferably of about 500 to about 5000.

According to a further embodiment, the nucleic acid sequence accordingto the invention is an artificial nucleic sequence as defined herein.

RNA:

In embodiments, the artificial nucleic acid is an RNA, in particular acircular RNA. As used herein, “circular RNA” has to be understood as acircular polynucleotide that can encode at least one antigenic peptideor protein as defined herein. Accordingly, in preferred embodiments,said circular RNA comprises at least one coding sequence encoding atleast one antigenic peptide or protein derived from a Henipavirus or afragment or variant thereof as defined herein.

The production of circRNAs can be performed using various methodsprovided in the art. For example, U.S. Pat. No. 6,210,931 teaches amethod of synthesizing circRNAs by inserting DNA fragments into aplasmid containing sequences having the capability of spontaneouscleavage and self-circularization. U.S. Pat. No. 5,773,244 teachesproducing circRNAs by making a DNA construct encoding an RNA cyclaseribozyme, expressing the DNA construct as an RNA, and then allowing theRNA to self-splice, which produces a circRNA free from intron in vitro.WO1992001813 teaches a process of making single strand circular nucleicacids by synthesizing a linear polynucleotide, combining the linearnucleotide with a complementary linking oligonucleotide underhybridization conditions, and ligating the linear polynucleotide. Theperson skilled in the art may also use methods provided in WO2015034925or WO2016011222 to produce circular RNA. Accordingly, methods forproducing circular RNA as provided in U.S. Pat. Nos. 6,210,931,5,773,244, WO1992001813, WO2015034925 and WO2016011222 are incorporatedherewith by reference.

In a preferred embodiment, the artificial nucleic acid is an RNA,preferably an mRNA.

The artificial RNA according to the present invention may be preparedusing any method known in the art, including chemical synthesis such ase.g. solid phase RNA synthesis, as well as in vitro methods, such as RNAin vitro transcription reactions.

In a preferred embodiment, the artificial nucleic acid as definedherein, preferably the RNA as defined herein, is obtained by RNA invitro transcription. Accordingly, the RNA of the invention is preferablyan in vitro transcribed RNA.

The terms “RNA in vitro transcription” or “in vitro transcription”relate to a process wherein RNA is synthesized in a cell-free system (invitro) as defined above. DNA, particularly plasmid DNA (or PCR product),is typically used as template for the generation of RNA transcripts.

In embodiments, the nucleotide mixture used in RNA in vitrotranscription may additionally contain modified nucleotides as definedherein. In embodiments, the nucleotide mixture (i.e. the fraction ofeach nucleotide in the mixture) may be optimized for the given RNAsequence, preferably as described WO2015/188933.

In embodiment where more than one different artificial nucleic acid asdefined herein has to be produced, e.g. where 2, 3, 4, 5, 6, 7, 8, 9, 10or even more different artificial nucleic acids have to be produced(e.g. encoding different antigenic peptides, proteins of Hendra and/orNipah virus), procedures as described in WO2017/109134 may be suitablyused.

In the context of nucleic acid vaccine production, it may be required toprovide GMP-grade RNA. GMP-grade RNA may be suitably produced using amanufacturing process approved by regulatory authorities. Accordingly,in a particularly preferred embodiment, RNA production is performedunder current good manufacturing practice (GMP), implementing variousquality control steps on DNA and RNA level, according to WO2016/180430.Accordingly, the RNA of the invention is a GMP-grade RNA, particularly aGMP-grade mRNA.

The obtained RNA products are preferably purified using PureMessenger®(CureVac, Tübingen, Germany; RP-HPLC according to WO2008/077592) and/ortangential flow filtration (as described in WO2016/193206).

In a preferred embodiment, the RNA, particularly the purified RNA, islyophilized according to WO2016/165831 or WO2011/069586 to yield atemperature stable dried artificial nucleic acid (powder) as definedherein. The RNA of the invention, particularly the purified RNA may alsobe dried using spray-drying or spray-freeze drying according toWO2016/184575 or WO2016184576 to yield a temperature stable artificialnucleic acid (powder) as defined herein. Accordingly, in the context ofmanufacturing and purifying nucleic acids, particularly RNA, thedisclosures of WO2017/109161, WO2015/188933, WO2016/180430,WO2008/077592, WO2016/193206, WO2016/165831, WO2011/069586,WO2016/184575, and WO2016/184576 are incorporated herewith by reference.

Accordingly, in preferred embodiments the RNA is a dried RNA,particularly a dried mRNA.

The term “dried RNA” as used herein has to be understood as RNA that hasbeen lyophilized, or spray-dried, or spray-freeze dried as defined aboveto obtain a temperature stable dried RNA (powder).

Accordingly, in preferred embodiments the RNA is a purified RNA,particularly purified mRNA.

The term “purified RNA” as used herein has to be understood as RNA whichhas a higher purity after certain purification steps (e.g. HPLC, TFF,precipitation steps) than the starting material (e.g. in vitrotranscribed RNA). Typical impurities that are essentially not present inpurified RNA comprise peptides or proteins (e.g. enzymes derived fromDNA dependent RNA in vitro transcription, e.g. RNA polymerases, RNases,BSA, pyrophosphatase, restriction endonuclease, DNase), spermidine,abortive RNA sequences, RNA fragments, free nucleotides (modifiednucleotides, conventional NTPs, cap analogue), plasmid DNA fragments,buffer components (HEPES, TRIS, MgCl₂) etc. Other impurities that may bederived from e.g. fermentation procedures comprise bacterial impurities(bioburden, bacterial DNA) or impurities derived from purificationprocedures (organic solvents etc.). Accordingly, it is desirable in thisregard for the “degree of RNA purity” to be as close as possible to100%. It is also desirable for the degree of RNA purity that the amountof full length RNA transcripts is as close as possible to 100%.Accordingly “purified RNA” as used herein has a degree of purity of morethan 70%, 75%, 80%, 85%, very particularly 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% and most favorably 99% or more. The degree of purity mayfor example be determined by an analytical HPLC, wherein the percentagesprovided above correspond to the ratio between the area of the peak forthe target RNA and the total area of all peaks representing theby-products. Alternatively, the degree of purity may for example bedetermined by an analytical agarose gel electrophoresis or capillary gelelectrophoresis.

It has to be understood that “dried RNA” as defined herein and “purifiedRNA” as defined herein or “GMP-grade mRNA” as defined herein may havesuperior stability characteristics and improved efficiency (e.g. bettertranslatability of the mRNA in vivo).

Nucleic Acid Modifications:

According to a further embodiment, the nucleic acid sequence accordingto the invention is a modified nucleic acid sequence, preferably amodified RNA sequence as described herein. In this context, amodification as defined herein preferably leads to a stabilization ofthe nucleic acid sequence according to the invention. More preferably,the invention thus provides a nucleic acid sequence, more preferably astabilized RNA sequence comprising at least one coding sequence asdefined herein.

According to one embodiment, the nucleic acid sequence of the presentinvention may thus be provided as a “stabilized nucleic acid sequence”,preferably as a “stabilized RNA sequence”, that is to say as an nucleicacid sequence or the RNA that is essentially resistant to in vivodegradation (e.g. by an exo- or endo-nuclease).

Such stabilization may be effected by providing a “dried RNA” and/or a“purified RNA” as specified herein. Alternatively or in addition tothat, such stabilization can be effected, for example, by a modifiedphosphate backbone of the nucleic acid, particularly of the RNA of thepresent invention. A backbone modification in connection with thepresent invention is a modification in which phosphates of the backboneof the nucleotides contained in the nucleic acid or the RNA arechemically modified. Nucleotides that may be preferably used in thisconnection contain e.g. a phosphorothioate-modified phosphate backbone,preferably at least one of the phosphate oxygens contained in thephosphate backbone being replaced by a sulfur atom. Stabilized nucleicacids or RNAs may further include, for example: non-ionic phosphateanalogues, such as, for example, alkyl and aryl phosphonates, in whichthe charged phosphonate oxygen is replaced by an alkyl or aryl group, orphosphodiesters and alkylphosphotriesters, in which the charged oxygenresidue is present in alkylated form. Such backbone modificationstypically include, without implying any limitation, modifications fromthe group consisting of methylphosphonates, phosphoramidates andphosphorothioates (e.g. cytidine-5′-O-(1-thiophosphate)).

In the following, specific modifications are described, which arepreferably capable of “stabilizing” the RNA as defined herein.

Chemical Modifications of Nucleic Acids:

The term “RNA modification” as used herein may refer to chemicalmodifications comprising backbone modifications as well as sugarmodifications or base modifications. In this context, a modified RNA(sequence) as defined herein may contain nucleotideanalogues/modifications, e.g. backbone modifications, sugarmodifications or base modifications. A backbone modification inconnection with the present invention is a modification, in whichphosphates of the backbone of the nucleotides contained in an RNA asdefined herein are chemically modified. A sugar modification inconnection with the present invention is a chemical modification of thesugar of the nucleotides of the RNA as defined herein. Furthermore, abase modification in connection with the present invention is a chemicalmodification of the base moiety of the nucleotides of the RNA. In thiscontext, nucleotide analogues or modifications are preferably selectedfrom nucleotide analogues, which are applicable for transcription and/ortranslation. The modified nucleosides and nucleotides, which may beincorporated into a modified nucleic acid or RNA as described herein,can be modified in the sugar moiety. For example, the 2′ hydroxyl group(OH) can be modified or replaced with a number of different “oxy” or“deoxy” substituents. Examples of “oxy”-2′ hydroxyl group modificationsinclude, but are not limited to, alkoxy or aryloxy (—OR, e.g., R═H,alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar);polyethyleneglycols (PEG), —O(CH2CH2O)nCH2CH2OR; “locked” nucleic acids(SNA) in which the 2′ hydroxyl is connected, e.g., by a methylenebridge, to the 4′ carbon of the same ribose sugar; and amino groups(—O-amino, wherein the amino group, e.g., NRR, can be alkylamino,dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, ordiheteroaryl amino, ethylene diamine, polyamino) or aminoalkoxy. “Deoxy”modifications include hydrogen, amino (e.g. NH2; alkylamino,dialkylamino, heterocyclyl, arylamino, diary) amino, heteroaryl amino,diheteroaryl amino, or amino acid); or the amino group can be attachedto the sugar through a linker, wherein the linker comprises one or moreof the atoms C, N, and O. The sugar group can also contain one or morecarbons that possess the opposite stereochemical configuration than thatof the corresponding carbon in ribose. Thus, a modified RNA can includenucleotides containing, for instance, arabinose as the sugar. Thephosphate backbone may further be modified in the modified nucleosidesand nucleotides, which may be incorporated into a modified nucleic acidor a modified RNA as described herein. The phosphate groups of thebackbone can be modified by replacing one or more of the oxygen atomswith a different substituent. Further, the modified nucleosides andnucleotides can include the full replacement of an unmodified phosphatemoiety with a modified phosphate as described herein. Examples ofmodified phosphate groups include, but are not limited to,phosphorothioate, phosphoroselenates, borano phosphates, boranophosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl oraryl phosphonates and phosphotriesters. Phosphorodithioates have bothnon-linking oxygens replaced by sulfur. The phosphate linker can also bemodified by the replacement of a linking oxygen with nitrogen (bridgedphosphoroamidates), sulfur (bridged phosphorothioates) and carbon(bridged methylene-phosphonates). The modified nucleosides andnucleotides, which may be incorporated into a modified nucleic acid orparticularly into a modified RNA as described herein, can further bemodified in the nucleobase moiety. Examples of nucleobases particularlyfound in RNA include, but are not limited to, adenine, guanine, cytosineand uracil. For example, the nucleosides and nucleotides describedherein can be chemically modified on the major groove face. In someembodiments, the major groove chemical modifications can include anamino group, a thiol group, an alkyl group, or a halo group. Inparticularly preferred embodiments of the present invention, thenucleotide analogues/modifications are selected from base modifications,which are preferably selected from2-amino-6-chloropurineriboside-5′-triphosphate,2-Aminopurine-riboside-5′-triphosphate;2-aminoadenosine-5′-triphosphate,2′-Amino-2′-deoxycytidine-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 2′-Fluorothymidine-5′-triphosphate,2′-O-Methyl-inosine-5′-triphosphate 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate,5-Bromo-2′-deoxycytidine-5″-triphosphate,5-Bromo-2′-deoxyuridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-lodo-2% deoxycytidine-5′-triphosphate, 5-iodouridine-5′-triphosphate,5-lodo-2′-deoxyuridine-5′-triphosphate,5-methylcytidine-5′-triphosphate, 5-methyluridine-5′-triphosphate,5-Propynyl-2′-deoxycytidine-5′-triphosphate,5-Propynyl-2′-deoxyuridine-5′-triphosphate,6-azacytidine-5′-triphosphate, 6-azauridine-5′-triphosphate,6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5″-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5″-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate. Insome embodiments, modified nucleosides include pyridin-4-oneribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine,4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine,3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine,5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine,1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine,1-taurinomethyl-4-thio-uridine, 5-methyl-uridine,1-methyl-pseudouridine, 4-thio-1-methyl-pseudouridine,2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine,dihydropseudouridine, 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine,4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine. In someembodiments, modified nucleosides include 5-aza-cytidine,pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine,5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine,1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine,4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine.In other embodiments, modified nucleosides include 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine,7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine,7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine,1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine,N6-(cis-hydroxyisopentenyl)adenosine,2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine,N6-glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine,2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine,7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine. In otherembodiments, modified nucleosides include inosine, 1-methyl-inosine,wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine,6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine,6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine,1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine,8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

In some embodiments, the nucleotide can be modified on the major grooveface and can include replacing hydrogen on C-5 of uracil with a methylgroup or a halo group. In specific embodiments, a modified nucleoside is5′-O-(1-thiophosphate)-adenosine, 5′-O-(1-thiophosphate)-cytidine,5′-O-(1-thiophosphate)-guanosine, 5′-O-(1-thiophosphate)-uridine or5′-O-(1-thiophosphate)-pseudouridine. In further specific embodiments,nucleoside modifications selected from 6-aza-cytidine, 2-thio-cytidine,α-thio-cytidine, Pseudo-iso-cytidine, 5-aminoallyl-uridine,5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine,α-thio-uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine,deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine,α-thio-guanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo-guanosine,7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine,N6-methyl-2-amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine,N6-methyl-adenosine, α-thio-adenosine, 8-azido-adenosine,7-deaza-adenosine.

Particularly preferred and suitable in the context of the invention arepseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and5-methoxyuridine. Accordingly, the artificial nucleic acid as definedherein may comprise at least one modified nucleotide selected frompseudouridine (ψ), N1-methylpseudouridine (m1ψ), 5-methylcytosine, and5-methoxyuridine.

According to a further embodiment, a modified nucleic acid, particularlya modified RNA as defined herein can contain a lipid modification. Sucha lipid-modified nucleic acid typically comprises a nucleic acid asdefined herein. Such a lipid-modified nucleic acid or RNA as definedherein typically further comprises at least one linker covalently linkedwith that nucleic acid or RNA, and at least one lipid covalently linkedwith the respective linker. Alternatively, the lipid-modified nucleicacid comprises at least one nucleic acid as defined herein and at leastone (bifunctional) lipid covalently linked (without a linker) with thatnucleic acid. According to a third alternative, the lipid-modifiednucleic acid comprises an nucleic acid molecule as defined herein, atleast one linker covalently linked with that RNA, and at least one lipidcovalently linked with the respective linker, and also at least one(bifunctional) lipid covalently linked (without a linker) with thatnucleic acid. In this context, it is particularly preferred that thelipid modification is present at the terminal ends of a linear nucleicacid sequence.

Sequence Modified Henipavirus Sequences:

According to preferred embodiments, the artificial nucleic acid of theinvention may be sequence-modified. Accordingly, in embodiments, the G/Ccontent of the at least one coding sequence is increased compared to theG/C content of the corresponding wild type coding sequence, and/or the Ccontent of the at least one coding sequence is increased compared to theC content of the corresponding wild type coding sequence and/or thecodons in the at least one coding sequence are adapted to human codonusage, and/or the codon adaptation index (CAI) is preferably increasedor maximised in the at least one coding sequence, wherein the amino acidsequence encoded by the at least one coding sequence is preferably notbeing modified compared to the amino acid sequence encoded by thecorresponding wild type coding sequence.

According to a preferred embodiment, the present invention provides anucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Henipavirus, wherein the at least one codingsequence comprises a (sequence modified) nucleic acid sequence, which isidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleicacid sequence selected from the group consisting of SEQ ID NOs: 53-234,625-806, 859-1040, 1093-1274, 1275-1508, 1519-1539 or a fragment orvariant of any of these sequences.

Sequence Modified Hendra Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprising at least one codingsequence, wherein the coding sequence comprises or consists of any oneof the (modified) RNA sequences as defined in the columns “C-J” of Table1 and Table 1B, or of a fragment or variant of any one of thesesequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (modified) RNA sequences as defined incolumns “C-J” of Table 1 and Table 1B, or of a fragment or variant ofany one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the nucleic acid sequence, encoding at least oneantigenic peptide or protein derived from Hendra virus, comprises orconsists of an RNA sequence having a sequence identity of at least 80%with any one of the (modified) RNA sequences as defined in the columns“C-J” of Table 1 and Table 1B, or of a fragment or variant of any one ofthese sequences.

Sequence Modified Nipah Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprising at least one codingsequence, wherein the coding sequence comprises or consists of any oneof the (modified) RNA sequences as defined in the columns “C-J” of Table2 and columns “C-J” of Table 2B, or of a fragment or variant of any oneof these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (modified) RNA sequences as defined incolumns “C-J” of Table 2 and in columns “C-J” of Table 2B, or of afragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the nucleic acid sequence, encoding at least oneantigenic peptide or protein derived from Nipah virus, comprises orconsists of an RNA sequence having a sequence identity of at least 80%with any one of the (modified) RNA sequences according as defined in thecolumns “C-J” of Table 2 and in columns “C-J” of Table 2B, or of afragment or variant of any one of these sequences.

G/C Content Modification:

According to an embodiment, the nucleic acid sequence of the presentinvention, may be modified, and thus stabilized, by modifying theguanosine/cytosine (G/C) content of the nucleic acid sequence,preferably of the at least one coding sequence of the nucleic acidsequence of the present invention.

In a particularly preferred embodiment of the present invention, the G/Ccontent of the coding sequence of the nucleic acid sequence of thepresent invention is modified, particularly increased, compared to theG/C content of the coding sequence of the respective wild type nucleicacid sequence, i.e. the unmodified nucleic acid. The amino acid sequenceencoded by the nucleic acid is preferably not modified as compared tothe amino acid sequence encoded by the respective wild type nucleicacid. This modification of the nucleic acid sequence of the presentinvention is based on the fact that the sequence of any nucleic acidregion, particularly the sequence of any RNA region to be translated isimportant for efficient translation of that nucleic acid, particularlyof that RNA. Thus, the composition of the nucleic acid and the sequenceof various nucleotides are important. In particular, in case of RNA,sequences having an increased G (guanosine)/C (cytosine) content aremore stable than sequences having an increased A (adenosine)/U (uracil)content. According to the invention, the codons of the nucleic acid aretherefore varied compared to the respective wild type nucleic acid,while retaining the translated amino acid sequence, such that theyinclude an increased amount of G/C nucleotides. In respect to the factthat several codons code for one and the same amino acid (so-calleddegeneration of the genetic code), the most favourable codons for thestability can be determined (so-called alternative codon usage).Depending on the amino acid to be encoded by the nucleic acid, there arevarious possibilities for modification of the nucleic acid sequence,compared to its wild type sequence.

The following modifications may apply for RNA molecules, but may also betransferrable to DNA molecules: In the case of amino acids, which areencoded by codons, containing exclusively G or C nucleotides, nomodification of the codon is necessary. Thus, the codons for Pro (CCC orCCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC or GGG) require nomodification, since no A or U is present. In contrast, codons whichcontain A and/or U nucleotides can be modified by substitution of othercodons, which code for the same amino acids but contain no A and/or U.Examples of these are: the codons for Pro can be modified from CCU orCCA to CCC or CCG; the codons for Arg can be modified from CGU or CGA orAGA or AGG to CGC or CGG; the codons for Ala can be modified from GCU orGCA to GCC or GCG; the codons for Gly can be modified from GGU or GGA toGGC or GGG. In other cases, although A or U nucleotides cannot beeliminated from the codons, it is however possible to decrease the A andU content by using codons which contain a lower content of A and/or Unucleotides. Examples of these are: the codons for Phe can be modifiedfrom UUU to UUC; the codons for Leu can be modified from UUA, UUG, CUUor CUA to CUC or CUG; the codons for Ser can be modified from UCU or UCAor AGU to UCC, UCG or AGC; the codon for Tyr can be modified from UAU toUAC; the codon for Cys can be modified from UGU to UGC; the codon forHis can be modified from CAU to CAC; the codon for Gln can be modifiedfrom CAA to CAG; the codons for Ile can be modified from AUU or AUA toAUC; the codons for Thr can be modified from ACU or ACA to ACC or ACG;the codon for Asn can be modified from AAU to AAC; the codon for Lys canbe modified from AAA to AAG; the codons for Val can be modified from GUUor GUA to GUC or GUG; the codon for Asp can be modified from GAU to GAC;the codon for Glu can be modified from GAA to GAG; the stop codon UAAcan be modified to UAG or UGA. In the case of the codons for Met (AUG)and Trp (UGG), on the other hand, there is no possibility of sequencemodification. The substitutions listed above can be used eitherindividually or in all possible combinations to increase the G/C contentof the RNA sequence of the present invention compared to its particularwild type RNA (i.e. the original sequence). Thus, for example, allcodons for Thr occurring in the wild type sequence can be modified toACC (or ACG). Preferably, however, for example, combinations of theabove substitution possibilities are used: substitution of all codonscoding for Thr in the original sequence (wild type RNA) to ACC (or ACG)and substitution of all codons originally coding for Ser to UCC (or UCGor AGC); substitution of all codons coding for Ile in the originalsequence to AUC and substitution of all codons originally coding for Lysto AAG and substitution of all codons originally coding for Tyr to UAC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) and substitution of all codons originally coding for Glu toGAG and substitution of all codons originally coding for Ala to GCC (orGCG) and substitution of all codons originally coding for Arg to CGC (orCGG); substitution of all codons coding for Val in the original sequenceto GUC (or GUG) and substitution of all codons originally coding for Gluto GAG and substitution of all codons originally coding for Ala to GCC(or GCG) and substitution of all codons originally coding for Gly to GGC(or GGG) and substitution of all codons originally coding for Asn to MC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) and substitution of all codons originally coding for Phe toUUC and substitution of all codons originally coding for Cys to UGC andsubstitution of all codons originally coding for Leu to CUG (or CUC) andsubstitution of all codons originally coding for Gln to CAG andsubstitution of all codons originally coding for Pro to CCC (or CCG);etc.

Preferably, the G/C content of the coding sequence of the RNA sequenceof the present invention is increased by at least 7%, more preferably byat least 15%, particularly preferably by at least 20%, compared to theG/C content of the coding sequence of the wild type RNA, which codes foran NIPAH virus antigen as defined herein or a fragment or variantthereof. According to a specific embodiment at least 5%, 10%, 20%, 30%,40%, 50%, 60%, more preferably at least 70%, even more preferably atleast 80% and most preferably at least 90%, 95% or even 100% of thesubstitutable codons in the region coding for a peptide or protein asdefined herein or a fragment or variant thereof or the whole sequence ofthe wild type RNA sequence are substituted, thereby increasing theGC/content of said sequence. In this context, it is particularlypreferable to increase the G/C content of the RNA sequence of thepresent invention, preferably of the at least one coding sequence of theRNA sequence according to the invention, to the maximum (i.e. 100% ofthe substitutable codons) as compared to the wild type sequence.According to the invention, a further preferred modification of the RNAsequence of the present invention is based on the finding that thetranslation efficiency is also determined by a different frequency inthe occurrence of tRNAs in cells. Thus, if so-called “rare codons” arepresent in the RNA sequence of the present invention to an increasedextent, the corresponding modified RNA sequence is translated to asignificantly poorer degree than in the case where codons coding forrelatively “frequent” tRNAs are present. According to the invention, inthe modified RNA sequence of the present invention, the region whichcodes for a peptide or protein as defined herein or a fragment orvariant thereof is modified compared to the corresponding region of thewild type RNA sequence such that at least one codon of the wild typesequence, which codes for a tRNA which is relatively rare in the cell,is exchanged for a codon, which codes for a tRNA which is relativelyfrequent in the cell and carries the same amino acid as the relativelyrare tRNA. By this modification, the sequence of the RNA of the presentinvention is modified such that codons for which frequently occurringtRNAs are available are inserted. In other words, according to theinvention, by this modification all codons of the wild type sequence,which code for a tRNA which is relatively rare in the cell, can in eachcase be exchanged for a codon, which codes for a tRNA which isrelatively frequent in the cell and which, in each case, carries thesame amino acid as the relatively rare tRNA. Which tRNAs occurrelatively frequently in the cell and which, in contrast, occurrelatively rarely is known to a person skilled in the art; cf. e.g.Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. The codons, whichuse for the particular amino acid the tRNA which occurs the mostfrequently, e.g. the Gly codon, which uses the tRNA, which occurs themost frequently in the (human) cell, are particularly preferred.According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe modified RNA sequence of the present invention, with the “frequent”codons without modifying the amino acid sequence of the protein encodedby the coding sequence of the RNA sequence. This preferred embodimentallows provision of a particularly efficiently translated and stabilized(modified) RNA sequence of the present invention. The determination of amodified RNA sequence of the present invention as described above(increased G/C content; exchange of tRNAs) can be carried out using thecomputer program explained in WO 02/098443—the disclosure content ofwhich is included in its full scope in the present invention. Using thiscomputer program, the nucleotide sequence of any desired RNA sequencecan be modified with the aid of the genetic code or the degenerativenature thereof such that a maximum G/C content results, in combinationwith the use of codons which code for tRNAs occurring as frequently aspossible in the cell, the amino acid sequence coded by the modified RNAsequence preferably not being modified compared to the non-modifiedsequence. Alternatively, it is also possible to modify only the G/Ccontent or only the codon usage compared to the original sequence. Thesource code in Visual Basic 6.0 (development environment used: MicrosoftVisual Studio Enterprise 6.0 with Servicepack 3) is also described in WO02/098443. In a further preferred embodiment of the present invention,the A/U content in the environment of the ribosome binding site of theRNA sequence of the present invention is increased compared to the A/Ucontent in the environment of the ribosome binding site of itsrespective wild type RNA. This modification (an increased A/U contentaround the ribosome binding site) increases the efficiency of ribosomebinding to the RNA. An effective binding of the ribosomes to theribosome binding site (Kozak sequence: SEQ ID NO: 255, 256; the AUGforms the start codon) in turn has the effect of an efficienttranslation of the RNA. According to a further embodiment of the presentinvention, the RNA sequence of the present invention may be modifiedwith respect to potentially destabilizing sequence elements.Particularly, the coding sequence and/or the 5′ and/or 3′ untranslatedregion of this RNA sequence may be modified compared to the respectivewild type RNA such that it contains no destabilizing sequence elements,the encoded amino acid sequence of the modified RNA sequence preferablynot being modified compared to its respective wild type RNA. It is knownthat, for example in sequences of eukaryotic RNAs, destabilizingsequence elements (DSE) occur, to which signal proteins bind andregulate enzymatic degradation of RNA in vivo. For further stabilizationof the modified RNA sequence, optionally in the region which encodes atleast one peptide or protein as defined herein or a fragment or variantthereof, one or more such modifications compared to the correspondingregion of the wild type RNA can therefore be carried out, so that no orsubstantially no destabilizing sequence elements are contained there.According to the invention, DSE present in the untranslated regions (3′-and/or 5′-UTR) can also be eliminated from the RNA sequence of thepresent invention by such modifications. Such destabilizing sequencesare e.g. AU-rich sequences (AURES), which occur in 3′-UTR sections ofnumerous unstable RNAs (Caput et al., Proc. Natl. Acad. Sci. USA 1986,83: 1670 to 1674). The RNA sequence of the present invention istherefore preferably modified compared to the respective wild type RNAsuch that the RNA sequence of the present invention contains no suchdestabilizing sequences. This also applies to those sequence motifswhich are recognized by possible endonucleases, e.g. the sequenceGAACAAG, which is contained in the 3′-UTR segment of the gene encodingthe transferrin receptor (Binder et al., EMBO J. 1994, 13: 1969 to1980). These sequence motifs are also preferably removed in the RNAsequence of the present invention.

G/C Content Modified Henipavirus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Henipavirus, wherein the at least one codingsequence comprises a (G/C modified) nucleic acid sequence, which isidentical or at least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleicacid sequence selected from the group consisting of any one of the(modified) RNA sequences as defined in the columns “C, G-J” (opt1, opt5,opt6, opt7) of Table 1, Table 1B, Table 2, and Table 2B.

According to a particularily preferred embodiment, the artificialnucleic acid according to the invention comprises at least one codingsequence, wherein the at least one coding sequence comprises a nucleicacid sequence, which is identical or at least 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%identical to a nucleic acid sequence selected from the group consistingof 53-78, 625-635, 859-869, 1093-1103, 636-650, 870-884, 1104-1118,1275-1508, 1519-1521 and as defined in columns “C” (opt1) of Table 1,Table 1B, Table 2, and Table 2B, or a fragment or variant of any ofthese sequences.

G/C Content Modified Hendra Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprising at least one codingsequence, wherein the coding sequence comprises or consists of any oneof the (G/C modified) RNA sequences as defined in columns “C, G-J”(opt1, opt5, opt6, opt7) of Table 1 and columns “C, G-J” (opt1, opt5,opt6, opt7) of Table 1B, or of a fragment or variant of any one of thesesequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus fusion protein (F), wherein the codingsequence comprises or consists of any one of the (G/C modified) RNAsequences according to SEQ ID NOs: 60-63, 164-167, 190-193, 216-219,632-635, 866-869, 1100-1103, 736-739, 970-973, 1204-1207, 762-765,996-999, 1230-1233, 788-791, 1022-1025, 1256-1259 and as defined incolumns “C, G-J” (opt1, opt5, opt6, opt7) of Table 1 and columns “C,G-J” (opt1, opt5, opt6, opt7) of Table 1B, or of a fragment or variantof any one of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (G/C modified) RNA sequencesaccording to SEQ ID NOs: 71-78, 175-182, 201-208, 227-234, 643-650,877-884, 1111-1118, 747-754, 981-988, 1215-1222, 773-780, 1007-1014,1241-1248, 799-806, 1033-1040, 1267-1274 and as defined in columns “C,G-J” (opt1, opt5, opt6, opt7) of Table 1 and columns “C, G-J” (opt1,opt5, opt6, opt7) of Table 1B, or of a fragment or variant of any one ofthese sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (G/C modified) RNA sequences as definedin columns “C, G-J” (opt1, opt5, opt6, opt7) of Table 1 and columns “C,G-J” (opt1, opt5, opt6, opt7) of Table 1B, or of a fragment or variantof any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Hendra virus, comprises or consists ofan nucleic acid sequence having a sequence identity of at least 80% withany one of the (G/C modified) RNA sequences according as defined incolumns “C, G-J” (“opt1, opt5, opt6, opt7”) of Table 1 and columns “C,G-J” (opt1, opt5, opt6, opt7) of Table 1B, or of a fragment or variantof any one of these sequences.

G/C Content Modified Nipah Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprising at least one codingsequence, wherein the coding sequence comprises or consists of any oneof the (G/C modified) RNA sequences as defined in columns “C, G-J”(opt1, opt5, opt6, opt7) of Table 2 and columns “C, G-J” (opt1, opt5,opt6, opt7) of Table 2B, or of a fragment or variant of any one of thesesequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus fusion protein (F), wherein the coding sequencecomprises or consists of any one of the (G/C modified) RNA sequencesaccording to SEQ ID NOs: 53-59, 157-163, 183-189, 209-215, 625-631,859-865, 1093-1099, 729-735, 963-969, 1197-1203, 755-761, 989-995,1223-1229, 781-787, 1015-1021, 1249-1255, 1519-1521, 1531-1539 and asdefined in columns “C, G-J” (opt1, opt5, opt6, opt7) of Table 2 andcolumns “C, G-J” (opt1, opt5, opt6, opt7) of Table 2B, or of a fragmentor variant of any one of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (G/C modified) RNA sequencesaccording to SEQ ID NOs: 64-70, 168-174, 194-200, 220-226, 636-642,870-876, 1104-1110, 740-746, 974-980, 1208-1214, 766-772, 1000-1006,1234-1240 and as defined in columns “C, G-J” (opt1, opt5, opt6, opt7) ofTable 2 and columns “C, G-J” (opt1, opt5, opt6, opt7) of Table 2B, or ofa fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (G/C modified) RNA sequences as definedin columns “C, G-J” (opt1, opt5, opt6, opt7) of Table 2 and columns “C,G-J” (opt1, opt5, opt6, opt7) of Table 2B, or of a fragment or variantof any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Nipah virus, according to the inventioncomprises or consists of an nucleic acid sequence having a sequenceidentity of at least 80% with any one of the (G/C modified) RNAsequences as defined in columns “C, G-J” (opt1, opt5, opt6, opt7) ofTable 2 and columns “C, G-J” (opt1, opt5, opt6, opt7) of Table 2B, or ofa fragment or variant of any one of these sequences.

Sequence Adaptation to Human Codon Usage:

According to the invention, a further preferred modification of thenucleic acid sequence of the present invention is based on the findingthat codons encoding the same amino acid typically occur at differentfrequencies. According to the invention, in the modified nucleic acidsequence of the present invention, the coding sequence as defined hereinis preferably modified compared to the corresponding coding sequence ofthe respective wild type nucleic acid such that the frequency of thecodons encoding the same amino acid corresponds to the naturallyoccurring frequency of that codon according to the human codon usage ase.g. shown in Table 4.

For example, in the case of the amino acid alanine (Ala) present in anamino acid sequence encoded by the at least one coding sequence of the anucleic acid sequence according to the invention, the wild type codingsequence is preferably adapted in a way that the codon “GCC” is usedwith a frequency of 0.40, the codon “GCT” is used with a frequency of0.28, the codon “GCA” is used with a frequency of 0.22 and the codon“GCG” is used with a frequency of 0.10 etc. (see Table 4).

TABLE 4 Human codon usage table Amino Amino acid codon fraction /1000acid codon fraction /1000 Ala GCG 0.10 7.4 Pro CCG 0.11 6.9 Ala GCA 0.2215.8 Pro CCA 0.27 16.9 Ala GCT 0.28 18.5 Pro CCT 0.29 17.5 Ala GCC* 0.4027.7 Pro CCC* 0.33 19.8 Cys TGT 0.42 10.6 Gln CAG* 0.73 34.2 Cys TGC*0.58 12.6 Gln CAA 0.27 12.3 Asp GAT 0.44 21.8 Arg AGG 0.22 12.0 Asp GAC*0.56 25.1 Arg AGA* 0.21 12.1 Glu GAG* 0.59 39.6 Arg CGG 0.19 11.4 GluGAA 0.41 29.0 Arg CGA 0.10 6.2 Phe TTT 0.43 17.6 Arg CGT 0.09 4.5 PheTTC* 0.57 20.3 Arg CGC 0.19 10.4 Gly GGG 0.23 16.5 Ser AGT 0.14 12.1 GlyGGA 0.26 16.5 Ser AGC* 0.25 19.5 Gly GGT 0.18 10.8 Ser TCG 0.06 4.4 GlyGGC* 0.33 22.2 Ser TCA 0.15 12.2 His CAT 0.41 10.9 Ser TCT 0.18 15.2 HisCAC* 0.59 15.1 Ser TCC 0.23 17.7 Ile ATA 0.14 7.5 Thr ACG 0.12 6.1 IleATT 0.35 16.0 Thr ACA 0.27 15.1 Ile ATC* 0.52 20.8 Thr ACT 0.23 13.1 LysAAG* 0.60 31.9 Thr ACC* 0.38 18.9 Lys AAA 0.40 24.4 Val GTG* 0.48 28.1Leu TTG 0.12 12.9 Val GTA 0.10 7.1 Leu TTA 0.06 7.7 Val GTT 0.17 11.0Leu CTG* 0.43 39.6 Val GTC 0.25 14.5 Leu CTA 0.07 7.2 Trp TGG* 1 13.2Leu CTT 0.12 13.2 Tyr TAT 0.42 12.2 Leu CTC 0.20 19.6 Tyr TAC* 0.58 15.3Met ATG* 1 22.0 Stop TGA* 0.61 1.6 Asn AAT 0.44 17.0 Stop TAG 0.17 0.8Asn AAC* 0.56 19.1 Stop TAA 0.22 1.0 *most frequent codon

Human Codon Usage Adapted Hendra Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Hendra virus,wherein the coding sequence comprises or consists of any one of the(human codon usage adapted) RNA sequences as defined in column “E”(opt3) of Table 1 and column “E” (opt3) of Table 1B, or of a fragment orvariant of any one of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus fusion protein (F), wherein the codingsequence comprises or consists of any one of the (human codon usageadapted) RNA sequences according to SEQ ID NOs: 112-115, 684-687,918-921, 1152-1155, and as defined in column “E” (opt3) of Table 1 andcolumn “E” (opt3) of Table 1B, or of a fragment or variant of any one ofthese sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (human codon usage adapted) RNAsequences according to SEQ ID NOs: 123-130, 695-702, 929-936, 1163-1170and as defined in column “E” (opt3) of Table 1 and column “E” (opt3) ofTable 1B, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (human codon usage adapted) RNAsequences as defined in column “E” (opt3) of Table 1 and column “E”(opt3) of Table 1B, or of a fragment or variant of any one of thesesequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Hendra virus, comprises or consists ofan nucleic acid sequence having a sequence identity of at least 80% withany one of the (human codon usage adapted) RNA sequences as defined incolumn “E” (opt3) of Table 1 and column “E” (opt3) of Table 1B, or of afragment or variant of any one of these sequences.

Human Codon Usage Adapted Nipah Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Nipah virus,wherein the coding sequence comprises or consists of any one of the(human codon usage adapted) RNA sequences as defined in column “E”(opt3) of Table 2 and column “E” (opt3) of Table 2B, or of a fragment orvariant of any one of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus fusion protein (F), wherein the coding sequencecomprises or consists of any one of the (human codon usage adapted) RNAsequences according to SEQ ID NOs: 105-111, 677-683, 911-917, 1145-1151,1525-1527 and as defined in column “E” (opt3) of Table 2 and column “E”(opt3) of Table 2B, or of a fragment or variant of any one of thesesequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (human codon usage adapted) RNAsequences according to SEQ ID NOs: 116-122, 688-694, 922-928, 1156-1162and as defined in column “E” (opt3) of Table 2 and column “E” (opt3) ofTable 2B, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (human codon usage adapted) RNAsequences as defined in column “E” (opt3) of Table 2 and column “E”(opt3) of Table 2B, or of a fragment or variant of any one of thesesequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Nipah virus, comprises or consists of annucleic acid sequence having a sequence identity of at least 80% withany one of the (human codon usage adapted) RNA sequences as defined incolumn “E” (opt3) of Table 2 and column “E” (opt3) of Table 2B, or of afragment or variant of any one of these sequences.

Codon-Optimization (CAI Maximization):

As described above it is preferred according to the invention, that allcodons of the wild type sequence which code for a tRNA, which isrelatively rare in the cell, are exchanged for a codon which codes for atRNA, which is relatively frequent in the cell and which, in each case,carries the same amino acid as the relatively rare tRNA. Therefore it isparticularly preferred that the most frequent codons are used for eachencoded amino acid (see Table 4, most frequent codons are marked withasterisks). Such an optimization procedure increases the codonadaptation index (CAI) and ultimately maximises the CAI. In the contextof the invention, sequences with increased or maximized CAI aretypically referred to as “codon-optimized” sequences and/or CAIincreased and/or maximized sequences. According to a preferredembodiment, the nucleic acid sequence of the present invention comprisesat least one coding sequence, wherein the coding sequence/sequence iscodon-optimized as described herein. More preferably, the codonadaptation index (CAI) of the at least one coding sequence is at least0.5, at least 0.8, at least 0.9 or at least 0.95. Most preferably, thecodon adaptation index (CAI) of the at least one coding sequence is 1.

For example, in the case of the amino acid alanine (Ala) present in theamino acid sequence encoded by the at least one coding sequence of thenucleic acid sequence according to the invention, the wild type codingsequence is adapted in a way that the most frequent human codon “GCC” isalways used for said amino acid, or for the amino acid Cysteine (Cys),the wild type sequence is adapted in a way that the most frequent humancodon “TGC” is always used for said amino acid etc.

Codon Optimized Hendra Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Hendra virus,wherein the coding sequence comprises or consists of any one of the(codon optimized) RNA sequences as defined in column “F” (opt4) of Table1 and column “F” (opt4) of Table 1B, or of a fragment or variant of anyone of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus fusion protein (F), wherein the codingsequence comprises or consists of any one of the (codon optimized) RNAsequences according to SEQ ID NOs: 138-141, 710-713, 944-947, 1178-1181and as defined in columns “F” (“opt4”) of Table 1 and columns “F”(“opt4”) of Table 1B, or of a fragment or variant of any one of thesesequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (codon optimized) RNA sequencesaccording to SEQ ID NOs: 149-156, 721-728, 955-962, 1189-1196 and asdefined in columns “F” (“opt4”) of Table 1 and columns “F” (“opt4”) ofTable 1B, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (codon optimized) RNA sequences asdefined in column “F” (opt4) of Table 1 and column “F” (opt4) of Table1B, or of a fragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Hendra virus, comprises or consists ofan nucleic acid sequence having a sequence identity of at least 80% withany one of the (codon optimized) RNA sequences as defined in column “F”(opt4) of Table 1 and column “F” (opt4) of Table 1B, or of a fragment orvariant of any one of these sequences.

Codon Optimized Nipah Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Nipah virus,wherein the coding sequence comprises or consists of any one of the(codon optimized) RNA sequences as defined in column “F” (opt4) of Table2 and column “F” (opt4) of Table 2B, or of a fragment or variant of anyone of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus fusion protein (F), wherein the coding sequencecomprises or consists of any one of the (codon optimized) RNA sequencesaccording to SEQ ID NOs: 131-137, 703-709, 937-943, 1171-1177, 1528-1530and as defined in column “F” (opt4) of Table 2 and column “F” (opt4) ofTable 2B, or of a fragment or variant of any one of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (codon optimized) RNA sequencesaccording to SEQ ID NOs: 142-148, 714-720, 948-954, 1182-1188 and asdefined in column “F” (opt4) of Table 2 and column “F” (opt4) of Table2B, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (codon optimized) RNA sequencesaccording as defined in column “F” (opt4) of Table 2 and column “F”(opt4) of Table 2B, or of a fragment or variant of any one of thesesequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Nipah virus, comprises or consists of annucleic acid sequence having a sequence identity of at least 80% withany one of the (codon optimized) RNA sequences as defined in column “F”(opt4) of Table 2 and column “F” (opt4) of Table 2B, or of a fragment orvariant of any one of these sequences.

Cytosine Optimization:

According to another embodiment, the nucleic acid sequence of thepresent invention may be modified by modifying, preferably increasing,the cytosine (C) content of the nucleic acid sequence, preferably of thecoding sequence of the nucleic acid sequence, more preferably the codingsequence of the RNA sequence.

In a particularly preferred embodiment of the present invention, the Ccontent of the coding sequence of the nucleic acid sequence of thepresent invention is modified, preferably increased, compared to the Ccontent of the coding sequence of the respective wild type nucleic acid,i.e. the unmodified nucleic acid. The amino acid sequence encoded by theat least one coding sequence of the nucleic acid sequence of the presentinvention is preferably not modified as compared to the amino acidsequence encoded by the respective wild type nucleic acid.

In a preferred embodiment of the present invention, the modified nucleicacid, particularly the modified RNA sequence is modified such that atleast 10%, 20%, 30%, 40%, 50%, 60%, 70% or 80%, or at least 90% of thetheoretically possible maximum cytosine-content or even a maximumcytosine-content is achieved.

In further preferred embodiments, at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or even 100% of the codons of the target nucleic acid,particularly the modified RNA wild type sequence, which are “cytosinecontent optimizable” are replaced by codons having a highercytosine-content than the ones present in the wild type sequence.

In a further preferred embodiment, some of the codons of the wild typecoding sequence may additionally be modified such that a codon for arelatively rare tRNA in the cell is exchanged by a codon for arelatively frequent tRNA in the cell, provided that the substitutedcodon for a relatively frequent tRNA carries the same amino acid as therelatively rare tRNA of the original wild type codon. Preferably, all ofthe codons for a relatively rare tRNA are replaced by a codon for arelatively frequent tRNA in the cell, except codons encoding aminoacids, which are exclusively encoded by codons not containing anycytosine, or except for glutamine (Gin), which is encoded by two codonseach containing the same number of cytosines.

In a further preferred embodiment of the present invention, the modifiedtarget nucleic acid, preferably the RNA is modified such that at least80%, or at least 90% of the theoretically possible maximumcytosine-content or even a maximum cytosine-content is achieved by meansof codons, which code for relatively frequent tRNAs in the cell, whereinthe amino acid sequence remains unchanged.

Due to the naturally occurring degeneracy of the genetic code, more thanone codon may encode a particular amino acid. Accordingly, 18 out of 20naturally occurring amino acids are encoded by more than one codon (withTryp and Met being an exception), e.g. by 2 codons (e.g. Cys, Asp, Glu),by three codons (e.g. Ile), by 4 codons (e.g. Al, Gly, Pro) or by 6codons (e.g. Leu, Arg, Ser). However, not all codons encoding the sameamino acid are utilized with the same frequency under in vivoconditions. Depending on each single organism, a typical codon usageprofile is established.

The term “cytosine content-optimizable codon” as used within the contextof the present invention refers to codons, which exhibit a lower contentof cytosines than other codons encoding the same amino acid.Accordingly, any wild type codon, which may be replaced by another codonencoding the same amino acid and exhibiting a higher number of cytosineswithin that codon, is considered to be cytosine-optimizable(C-optimizable). Any such substitution of a C-optimizable wild typecodon by the specific C-optimized codon within a wild type codingsequence increases its overall C-content and reflects a C-enrichedmodified nucleic acid sequence. According to a preferred embodiment, thenucleic acid sequence, particularly the RNA sequence of the presentinvention, preferably the at least one coding sequence of the nucleicacid sequence of the present invention comprises or consists of aC-maximized RNA sequence containing C-optimized codons for allpotentially C-optimizable codons. Accordingly, 100% or all of thetheoretically replaceable C-optimizable codons are preferably replacedby C-optimized codons over the entire length of the coding sequence.

In this context, cytosine-content optimizable codons are codons, whichcontain a lower number of cytosines than other codons coding for thesame amino acid. Any of the codons GCG, GCA, GCU codes for the aminoacid Ala, which may be exchanged by the codon GCC encoding the sameamino acid, and/or the codon UGU that codes for Cys may be exchanged bythe codon UGC encoding the same amino acid, and/or the codon GAU whichcodes for Asp may be exchanged by the codon GAC encoding the same aminoacid, and/or the codon that UUU that codes for Phe may be exchanged forthe codon UUC encoding the same amino acid, and/or any of the codonsGGG, GGA, GGU that code Gly may be exchanged by the codon GGC encodingthe same amino acid, and/or the codon CAU that codes for His may beexchanged by the codon CAC encoding the same amino acid, and/or any ofthe codons AUA, AUU that code for Ile may be exchanged by the codon AUC,and/or any of the codons UUG, UUA, CUG, CUA, CUU coding for Leu may beexchanged by the codon CUC encoding the same amino acid, and/or thecodon AAU that codes for Asn may be exchanged by the codon AAC encodingthe same amino acid, and/or any of the codons CCG, CCA, CCU coding forPro may be exchanged by the codon CCC encoding the same amino acid,and/or any of the codons AGG, AGA, CGG, CGA, CGU coding for Arg may beexchanged by the codon CGC encoding the same amino acid, and/or any ofthe codons AGU, AGC, UCG, UCA, UCU coding for Ser may be exchanged bythe codon UCC encoding the same amino acid, and/or any of the codonsACG, ACA, ACU coding for Thr may be exchanged by the codon ACC encodingthe same amino acid, and/or any of the codons GUG, GUA, GUU coding forVal may be exchanged by the codon GUC encoding the same amino acid,and/or the codon UAU coding for Tyr may be exchanged by the codon UACencoding the same amino acid.

In any of the above instances, the number of cytosines is increased by 1per exchanged codon. Exchange of all non C-optimized codons(corresponding to C-optimizable codons) of the coding sequence resultsin a C-maximized coding sequence. In the context of the invention, atleast 70%, preferably at least 80%, more preferably at least 90%, of thenon C-optimized codons within the at least one coding sequence of theRNA sequence according to the invention are replaced by C-optimizedcodons.

It may be preferred that for some amino acids the percentage ofC-optimizable codons replaced by C-optimized codons is less than 70%,while for other amino acids the percentage of replaced codons is higherthan 70% to meet the overall percentage of C-optimization of at least70% of all C-optimizable wild type codons of the coding sequence.

Preferably, in a C-optimized RNA sequence of the invention, at least 50%of the C-optimizable wild type codons for any given amino acid arereplaced by C-optimized codons, e.g. any modified C-enriched RNAsequence preferably contains at least 50% C-optimized codons atC-optimizable wild type codon positions encoding any one of the abovementioned amino acids Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Pro,Arg, Ser, Thr, Val and Tyr, preferably at least 60%.

In this context codons encoding amino acids, which are not cytosinecontent-optimizable and which are, however, encoded by at least twocodons, may be used without any further selection process. However, thecodon of the wild type sequence that codes for a relatively rare tRNA inthe cell, e.g. a human cell, may be exchanged for a codon that codes fora relatively frequent tRNA in the cell, wherein both code for the sameamino acid. Accordingly, the relatively rare codon GAA coding for Glumay be exchanged by the relative frequent codon GAG coding for the sameamino acid, and/or the relatively rare codon AAA coding for Lys may beexchanged by the relative frequent codon AAG coding for the same aminoacid, and/or the relatively rare codon CAA coding for Gln may beexchanged for the relative frequent codon CAG encoding the same aminoacid.

In this context, the amino acids Met (AUG) and Trp (UGG), which areencoded by only one codon each, remain unchanged. Stop codons are notcytosine-content optimized, however, the relatively rare stop codonsamber, ochre (UAA, UAG) may be exchanged by the relatively frequent stopcodon opal (UGA).

The single substitutions listed above may be used individually as wellas in all possible combinations in order to optimize thecytosine-content of the modified nucleic acid sequence compared to thewild type nucleic acid sequence.

Accordingly, the at least one coding sequence as defined herein may bechanged compared to the coding sequence of the respective wild typenucleic acid in such a way that an amino acid encoded by at least two ormore codons, of which one comprises one additional cytosine, such acodon may be exchanged by the C-optimized codon comprising oneadditional cytosine, wherein the amino acid is preferably unalteredcompared to the wild type sequence.

C-Optimized Hendra Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Hendra virus,wherein the coding sequence comprises or consists of any one of the(C-optimized) RNA sequences as defined in column “D” (“opt2”) of Table 1and in column “D” (opt2) of Table 1B, or of a fragment or variant of anyone of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus fusion protein (F), wherein the codingsequence comprises or consists of any one of the (C-optimized) RNAsequences according to SEQ ID NOs: 86-89, 658-661, 892-895, 1126-1129and as defined in column “D” (“opt2”) of Table 1 and in column “D”(opt2) of Table 1B, or of a fragment or variant of any one of thesesequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Hendra virus antigenic peptide or proteinderived from Hendra virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (C-optimized) RNA sequencesaccording to SEQ ID NOs: 97-104, 669-676, 903-910, 1137-1144 and asdefined in column “D” (“opt2”) of Table 1 and in column “D” (opt2) ofTable 1B, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (C-optimized) RNA sequences as definedin column “D” (“opt2”) of Table 1 and in column “D” (opt2) of Table 1B,or of a fragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Hendra virus, comprises or consists ofan nucleic acid sequence having a sequence identity of at least 80% withany one of the (C-optimized) RNA sequences as defined in column “D”(“opt2”) of Table 1 and in column “D” (opt2) of Table 1B, or of afragment or variant of any one of these sequences.

C-Optimized Nipah Virus Sequences:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Nipah virus,wherein the coding sequence comprises or consists of any one of the(C-optimized) RNA sequences as defined in column “D” (opt2) of Table 2and in column “D” (opt2) of Table 2B, or of a fragment or variant of anyone of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus fusion protein (F), wherein the coding sequencecomprises or consists of any one of the (C-optimized) RNA sequencesaccording to SEQ ID NOs: 79-85, 651-657, 885-891, 1119-1125, 1522-1524and as defined in column “D” (opt2) of Table 2 and in column “D” (opt2)of Table 2B, or of a fragment or variant of any one of these sequences.

According to a preferred embodiment, the present invention provides annucleic acid sequence as defined herein comprising at least one codingsequence encoding at least one Nipah virus antigenic peptide or proteinderived from Nipah virus glycoprotein (G), wherein the coding sequencecomprises or consists of any one of the (C-optimized) RNA sequencesaccording to SEQ ID NOs: 90-96, 662-668, 896-902, 1130-1136 and asdefined in column “D” (opt2) of Table 2 and in column “D” (opt2) ofTable 2B, or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Nipah virus, comprises or consists of an nucleicacid sequence identical to or having a sequence identity of at least 5%,10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least70%, more preferably of at least 80%, even more preferably at least 85%,even more preferably of at least 90% and most preferably of at least 95%or even 97%, with any one of the (C-optimized) RNA sequences as definedin column “D” (opt2) of Table 2 and in column “D” (opt2) of Table 2B, orof a fragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the RNA sequence, encoding at least one antigenicpeptide or protein derived from Nipah virus, comprises or consists of annucleic acid sequence having a sequence identity of at least 80% withany one of the (C-optimized) RNA sequences as defined in column “D”(opt2) of Table 2 and in column “D” (opt2) of Table 2B, or of a fragmentor variant of any one of these sequences.

Sequence Modified Secretory Signal Peptides:

According to a preferred embodiment, the present invention provides anucleic acid sequence comprising at least one coding sequence, encodingat least one antigenic peptide or protein derived from Hendra virusand/or Nipah virus, and, additionally, an N-terminal secretory signalpeptide, wherein the coding sequence comprises or consists of any one ofthe (modified) RNA sequences as defined in the columns “C-J” of Table 3,or of a fragment or variant of any one of these sequences.

In a further preferred embodiment, the at least one coding sequence ofthe nucleic acid sequence, encoding at least one antigenic peptide orprotein derived from Hendra virus and/or Nipah virus, and, additionally,an N-terminal secretory signal peptide, wherein the coding sequencecomprises or consists of an nucleic acid sequence identical to or havinga sequence identity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, or 99%, preferably of at least 70%, more preferably of at least80%, even more preferably at least 85%, even more preferably of at least90% and most preferably of at least 95% or even 97%, with any one of the(modified) RNA sequences as defined in the columns “C-J” of Table 3, orof a fragment or variant of any one of these sequences.

According to a particularly preferred embodiment, the at least onecoding sequence of the nucleic acid sequence comprises or consists of anRNA sequence having a sequence identity of at least 80% with any one ofthe (modified) RNA sequences as defined in the columns “C-J” of Table 3,or of a fragment or variant of any one of these sequences.

According to preferred embodiments, the present invention provides anucleic acid sequence, wherein the at least one coding sequencecomprises a (G/C modified) nucleic acid sequence, which is identical orat least 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleic acidsequence selected from the group consisting of any one of the (modified)RNA sequences as defined in the columns “C, G-J” (opt1, opt5, opt6,opt7) of Table 3.

5′-Cap Structure:

According to another preferred embodiment of the invention, a modifiednucleic acid sequence as defined herein, particularly a modified RNA asdefined herein can be modified by the addition of a so-called “5′-cap”structure, which preferably stabilizes the nucleic acid as describedherein. A 5′-cap is an entity, typically a modified nucleotide entity,which generally “caps” the 5′-end of a mature RNA. A 5′-cap maytypically be formed by a modified nucleotide, particularly by aderivative of a guanine nucleotide. Preferably, the 5′-cap is linked tothe 5′-terminus via a 5′-5′-triphosphate linkage. A 5′-cap may bemethylated, e.g. m7GpppN, wherein N is the terminal 5′ nucleotide of thenucleic acid carrying the 5′-cap, typically the 5′-end of an RNA.m7GpppN is the 5′-cap structure, which naturally occurs in RNAtranscribed by polymerase II and is therefore preferably not consideredas modification comprised in a modified RNA in this context.Accordingly, a modified RNA sequence of the present invention maycomprise a m7GpppN as 5′-cap, but additionally the modified RNA sequencetypically comprises at least one further modification as defined herein.

Further examples of 5′-cap structures include glyceryl, inverted deoxyabasic residue (moiety), 4′,5′ methylene nucleotide,1-(beta-D-erythrofuranosyl) nucleotide, 4′-thio nucleotide, carbocyclicnucleotide, 1,5-anhydrohexitol nucleotide, L-nucleotides,alpha-nucleotide, modified base nucleotide, threo-pentofuranosylnucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutylnucleotide, acyclic 3,5 dihydroxypentyl nucleotide, 3′-3′-invertednucleotide moiety, 3′-3′-inverted abasic moiety, 3′-2′-invertednucleotide moiety, 3% 2′-inverted abasic moiety, 1,4-butanediolphosphate, 3′-phosphoramidate, hexylphosphate, aminohexyl phosphate,3′-phosphate, 3′phosphorothioate, phosphorodithioate, or bridging ornon-bridging methylphosphonate moiety. These modified 5′-cap structuresare regarded as at least one modification in this context.

Particularly preferred modified 5′-cap structures are cap1 (methylationof the ribose of the adjacent nucleotide of m7G), cap2 (additionalmethylation of the ribose of the 2nd nucleotide downstream of the m7G),cap3 (additional methylation of the ribose of the 3rd nucleotidedownstream of the m7G), cap4 (methylation of the ribose of the 4thnucleotide downstream of the m7G), ARCA (anti-reverse cap analogue,modified ARCA (e.g. phosphothioate modified ARCA), inosine,N1-methyl-guanosine, 2′-fluoro-guanosine, 7-deaza-guanosine,8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and2-azido-guanosine. Accordingly, the RNA according to the inventionpreferably comprises a 5′-cap structure.

In a preferred embodiment, the 5′-cap structure is addedco-transcriptionally using cap-analogues as defined herein in an RNA invitro transcription reaction as defined herein. In another embodiment,the 5′-cap structure is added via enzymatic capping using cappingenzymes (e.g. vaccinia virus capping enzymes). In another embodiment,the 5′-cap structure is added via enzymatic capping using immobilizedcapping enzymes, e.g. in a capping reactor (WO 2016/193226).

Accordingly, in preferred embodiments, the artificial nucleic acid ofthe invention comprises a 5′-cap structure as defined herein.

Poly(A) Sequence/Tail:

According to a further preferred embodiment, the nucleic acid sequence,particularly the RNA sequence of the present invention may contain apoly-A tail on the 3′-terminus of typically about 10 to 200 adenosinenucleotides, preferably about 10 to 100 adenosine nucleotides, morepreferably about 40 to 80 adenosine nucleotides or even more preferablyabout 50 to 70 adenosine nucleotides.

Preferably, the poly(A) sequence in the RNA sequence of the presentinvention is derived from a DNA template by RNA in vitro transcription.Alternatively, the poly(A) sequence may also be obtained in vitro bycommon methods of chemical-synthesis without being necessarilytranscribed from a DNA-progenitor. Moreover, poly(A) sequences, orpoly(A) tails may be generated by enzymatic polyadenylation of the RNAaccording to the present invention using commercially availablepolyadenylation kits and corresponding protocols known in the art, orusing immobilized poly(A)polymerases e.g. in a polyadenylation reactor(WO/2016/174271)

Alternatively, the RNA as described herein optionally comprises apolyadenylation signal, which is defined herein as a signal, whichconveys polyadenylation to a (transcribed) RNA by specific proteinfactors (e.g. cleavage and polyadenylation specificity factor (CPSF),cleavage stimulation factor (CstF), cleavage factors I and II (CF I andCF II), poly(A) polymerase (PAP)). In this context, a consensuspolyadenylation signal is preferred comprising the NN(U/T)ANA consensussequence. In a particularly preferred aspect, the polyadenylation signalcomprises one of the following sequences: AA(U/T)AAA or A(U/T)(U/T)AAA(wherein uridine is usually present in RNA and thymidine is usuallypresent in DNA).

Poly(C) Sequence:

According to a further preferred embodiment, the nucleic acid sequence,particularly the RNA sequence of the present invention may contain apoly(C) tail on the 3′-terminus of typically about 10 to 200 cytosinenucleotides, preferably about 10 to 100 cytosine nucleotides, morepreferably about 20 to 70 cytosine nucleotides or even more preferablyabout 20 to 60 or even 10 to 40 cytosine nucleotides. Preferably, thepoly(C) sequence in the RNA sequence of the present invention is derivedfrom a DNA template by RNA in vitro transcription.

UTRs:

In a preferred embodiment, the artificial nucleic acid of the inventioncomprises at least one untranslated region (UTR.

In a preferred embodiment, the nucleic acid sequence, particularly theRNA sequence according to the invention comprises at least one 5′-and/or 3′-UTR element. In this context, an UTR element comprises orconsists of a nucleic acid sequence, which is derived from the 5′- or3′-UTR of any naturally occurring gene or which is derived from afragment, a homolog or a variant of the 5′- or 3′-UTR of a gene.Preferably, the 5′- or 3′-UTR element used according to the presentinvention is heterologous to the at least one coding sequence of the RNAsequence of the invention. Even if 5′- or 3′-UTR elements derived fromnaturally occurring genes are preferred, also synthetically engineeredUTR elements may be used in the context of the present invention.

3′-UTR Elements:

In preferred embodiment, the artificial nucleic acid of the inventioncomprises at least one 3′-UTR.

In a particularly preferred embodiment, the artificial nucleic acid ofthe invention comprises at least one heterologous 3′-UTR.

Preferably, the 3′-UTR comprises a poly(A) sequence and/or a poly(C)sequence as defined above, wherein the poly(A) sequence comprises 10 to200, 10 to 100, 40 to 200, 40 to 80 or 50 to 70 adenosine nucleotides,and/or the poly(C) sequence comprises 10 to 200, 10 to 100, 20 to 70, 20to 60 or 10 to 40 cytosine nucleotides.

The term “3′-UTR element” typically refers to a nucleic acid sequence,which comprises or consists of a nucleic acid sequence that is derivedfrom a 3′-UTR or from a variant of a 3′-UTR. A 3′-UTR element in thesense of the present invention may represent the 3′-UTR of a nucleicacid molecule, particularly of an RNA or DNA, preferably an mRNA. Thus,in the sense of the present invention, preferably, a 3′-UTR element maybe the 3′-UTR of an RNA, preferably of an mRNA, or it may be thetranscription template for a 3′-UTR of an RNA. Thus, a 3′-UTR elementpreferably is a nucleic acid sequence which corresponds to the 3′-UTR ofan RNA, preferably to the 3′-UTR of an mRNA, such as an mRNA obtained bytranscription of a genetically engineered vector construct. Preferably,the 3′-UTR element fulfils the function of a 3′-UTR or encodes asequence which fulfils the function of a 3′-UTR.

Preferably, the at least one 3′-UTR element comprises or consists of anucleic acid sequence derived from the 3′-UTR of a chordate gene,preferably a vertebrate gene, more preferably a mammalian gene, mostpreferably a human gene, or from a variant of the 3′-UTR of a chordategene, preferably a vertebrate gene, more preferably a mammalian gene,most preferably a human gene.

Preferably, the nucleic acid sequence, particularly the RNA sequence ofthe present invention comprises a 3′-UTR element, which may be derivablefrom a gene that relates to an RNA with an enhanced half-life (thatprovides a stable RNA), for example a 3′-UTR element as defined anddescribed below. Preferably, the 3′-UTR element is a nucleic acidsequence derived from a 3′ UTR of a gene, which preferably encodes astable RNA, or from a homolog, a fragment or a variant of said gene.

In a particularly preferred embodiment, the 3′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 3′-UTR of agene selected from the group consisting of an albumin gene, an α-globingene, a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene,and a collagen alpha gene, such as a collagen alpha 1(1) gene, or from avariant of a 3′-UTR of a gene selected from the group consisting of analbumin gene, an α-globin gene, a β-globin gene, a tyrosine hydroxylasegene, a lipoxygenase gene, and a collagen alpha gene, such as a collagenalpha 1(1) gene according to SEQ ID NOs: 1369-1390 of the patentapplication WO2013/143700, whose disclosure is incorporated herein byreference, or from a homolog, a fragment or a variant thereof. In aparticularly preferred embodiment, the 3′-UTR element comprises orconsists of a nucleic acid sequence which is derived from a 3′-UTR of analbumin gene, preferably a vertebrate albumin gene, more preferably amammalian albumin gene, most preferably a human albumin gene accordingto SEQ ID NOs: 247, 249, 251 or the corresponding RNA sequence SEQ IDNOs: 248, 250, 252.

In this context it is particularly preferred that the RNA sequenceaccording to the invention comprises a 3′-UTR element comprising acorresponding RNA sequence derived from the nucleic acids according toSEQ ID NOs: 1369-1390 of the patent application WO2013/143700 or afragment, homolog or variant thereof.

Most preferably the 3′-UTR element comprises the nucleic acid sequencederived from a fragment of the human albumin gene according to SEQ IDNOs: 249-252.

In this context, it is particularly preferred that the 3′-UTR element ofthe RNA sequence according to the present invention comprises orconsists of a corresponding RNA sequence of the nucleic acid sequenceaccording to SEQ ID NOs: 249 or 251 as shown in SEQ ID NOs: 250 or 252.

In another particularly preferred embodiment, the 3′-UTR elementcomprises or consists of a nucleic acid sequence which is derived from a3′-UTR of an alpha- or beta-globin gene, preferably a vertebrate alpha-or beta-globin gene, more preferably a mammalian alpha- or beta-globingene, most preferably a human alpha- or beta-globin gene according toSEQ ID NOs: 239, 241, 243, 245 or the corresponding RNA sequences SEQ IDNOs: 240, 242, 244, 246.

For example, the 3′-UTR element may comprise or consist of the center,α-complex-binding portion of the 3′-UTR of an α-globin gene, such as ofa human α-globin gene, or a homolog, a fragment, or a variant of anα-globin gene, preferably according to SEQ ID NO: 245 or 246.

In this context it is particularly preferred that the 3′-UTR element ofthe RNA sequence according to the invention comprises or consists of acorresponding RNA sequence of the nucleic acid sequence according to SEQID NO: 245 as shown in SEQ ID NO: 246, or a homolog, a fragment orvariant thereof.

The term “a nucleic acid sequence which is derived from the 3′-UTR of a[ . . . ] gene” preferably refers to a nucleic acid sequence which isbased on the 3′-UTR sequence of a [ . . . ] gene or on a part thereof,such as on the 3′-UTR of an albumin gene, an alpha-globin gene, abeta-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or acollagen alpha gene, such as a collagen alpha 1(1) gene, preferably ofan albumin gene or on a part thereof. This term includes sequencescorresponding to the entire 3′-UTR sequence, i.e. the full length 3′-UTRsequence of a gene, and sequences corresponding to a fragment of the3′-UTR sequence of a gene, such as an albumin gene, alpha-globin gene,beta-globin gene, tyrosine hydroxylase gene, lipoxygenase gene, orcollagen alpha gene, such as a collagen alpha 1(1) gene, preferably ofan albumin gene.

The term “a nucleic acid sequence which is derived from a variant of the3′-UTR of a [ . . . ] gene” preferably refers to a nucleic acidsequence, which is based on a variant of the 3′-UTR sequence of a gene,such as on a variant of the 3′-UTR of an albumin gene, an α-globin gene,a β-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, or acollagen alpha gene, such as a collagen alpha 1(I) gene, or on a partthereof as described above. This term includes sequences correspondingto the entire sequence of the variant of the 3′-UTR of a gene, i.e. thefull length variant 3′-UTR sequence of a gene, and sequencescorresponding to a fragment of the variant 3′-UTR sequence of a gene. Afragment in this context preferably consists of a continuous stretch ofnucleotides corresponding to a continuous stretch of nucleotides in thefull-length variant 3′-UTR, which represents at least 20%, preferably atleast 30%, more preferably at least 40%, more preferably at least 50%,even more preferably at least 60%, even more preferably at least 70%,even more preferably at least 80%, and most preferably at least 90% ofthe full-length variant 3′-UTR. Such a fragment of a variant, in thesense of the present invention, is preferably a functional fragment of avariant as described herein.

In further embodiments, the artificial nucleic acid as defined herein,particularly the RNA as defined herein comprises a 3′-UTR element, whichmay be any 3′-UTR element described in WO2016/107877. In this context,the disclosure of WO2016/107877 relating to 3′-UTR elements/sequences isherewith incorporated by reference. Particularly preferred 3′-UTRelements are SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318 of the patentapplication WO2016/107877, or fragments or variants of these sequences.In this context, it is particularly preferred that the 3′-UTR element ofthe RNA sequence according to the present invention comprises orconsists of a corresponding RNA sequence of the nucleic acid sequenceaccording SEQ ID NOs: 1 to 24 and SEQ ID NOs: 49 to 318 of the patentapplication WO2016/107877.

In embodiments, the artificial nucleic acid as defined herein,particularly the RNA as defined herein comprises a 3′-UTR element, whichmay be any 3′-UTR element as described in WO2017/036580. In thiscontext, the disclosure of WO2017/036580 relating to 3′-UTRelements/sequences is herewith incorporated by reference. Particularlypreferred 3′-UTR elements are nucleic acid sequences according to SEQ IDNOs: 152 to 204 of the patent application WO2017/036580, or fragments orvariants of these sequences. In this context, it is particularlypreferred that the 3′-UTR element of the RNA sequence according to thepresent invention comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according SEQ ID NOs: 152 to 204 of thepatent application WO2017/036580.

According to a preferred embodiment, the nucleic acid sequence,particularly the RNA sequence according to the invention comprises a5′-cap structure and/or at least one 3′-untranslated region element(3′-UTR element), preferably as defined herein. More preferably, the RNAfurther comprises a 5′-UTR element as defined herein.

In a preferred embodiment the RNA sequence comprises, preferably in 5′-to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b1.) optionally, at least one coding sequence encoding at least one    heterologous secretory signal sequence;-   b2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Henipavirus, or a fragment or    variant thereof,-   c.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from an alpha globin gene, preferably    comprising the corresponding RNA sequence of the nucleic acid    sequence according to SEQ ID NO: 246, a homolog, a fragment or a    variant thereof;-   d.) optionally, a poly(A) sequence, preferably comprising 64    adenosines;-   e.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;

In a particularly preferred embodiment the RNA sequence comprises,preferably in 5′- to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b1.) optionally, at least one coding sequence encoding at least one    heterologous secretory signal sequence, preferably selected from SEQ    ID NOs 317-572;-   b2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Hendra virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid sequences according to SEQ ID NOs:    34-37, 45-52, 60-63, 71-78, 86-89, 97-104, 112-115, 123-130,    138-141, 149-156, 164-167, 175-182, 190-193, 201-208, 216-219,    227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739,    762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728,    747-754, 773-780, 799-806, 840-843, 866-869, 892-895, 918-921,    944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884, 903-910,    929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077,    1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233,    1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196,    1215-1222, 1241-1248, 1267-1274, or a fragment or variant thereof;-   c.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from an alpha globin gene, preferably    comprising the corresponding RNA sequence of the nucleic acid    sequence according to SEQ ID NO: 246, a homolog, a fragment or a    variant thereof;-   d.) optionally, a poly(A) sequence, preferably comprising 64    adenosines;-   e.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;

In a further particularly preferred embodiment the RNA sequencecomprises, preferably in 5′- to 3′-direction:

-   a) a 5′-cap structure, preferably m7GpppN;-   b1.) optionally, at least one coding sequence encoding at least one    heterologous secretory signal sequence, preferably selected from SEQ    ID NOs 317-572;-   b2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Nipah virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid sequences according to SEQ ID NOs:    27-33, 38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137,    142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226,    599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761,    781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,    766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943,    963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928,    948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099,    1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,    1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214,    1234-1240, 1260-1266, 1516-1539 or a fragment or variant thereof;-   c.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from an alpha globin gene, preferably    comprising the corresponding RNA sequence of the nucleic acid    sequence according to SEQ ID NO: 246, a homolog, a fragment or a    variant thereof;-   d.) optionally, a poly(A) sequence, preferably comprising 64    adenosines;-   e.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;

5′-UTR Elements:

In a particularly preferred embodiment, the at least one nucleic acidsequence, in particular, the RNA sequence comprises at least one5′-untranslated region element (5′-UTR element). Preferably, the atleast one 5′-UTR element comprises or consists of a nucleic acidsequence, which is derived from the 5′-UTR of a TOP gene or which isderived from a fragment, homolog or variant of the 5′-UTR of a TOP gene.

It is particularly preferred that the 5′-UTR element does not comprise aTOP-motif or a 5′-TOP, as defined above.

In some embodiments, the nucleic acid sequence of the 5′-UTR element,which is derived from a 5′-UTR of a TOP gene, terminates at its 3′-endwith a nucleotide located at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10upstream of the start codon (e.g. A(U/T)G) of the gene or RNA it isderived from. Thus, the 5′-UTR element does not comprise any part of theprotein coding sequence. Thus, preferably, the only protein coding partof the at least one nucleic acid sequence, particularly of the RNAsequence, is provided by the coding sequence.

The nucleic acid sequence derived from the 5′-UTR of a TOP gene ispreferably derived from a eukaryotic TOP gene, preferably a plant oranimal TOP gene, more preferably a chordate TOP gene, even morepreferably a vertebrate TOP gene, most preferably a mammalian TOP gene,such as a human TOP gene.

For example, the 5′-UTR element is preferably selected from 5′-UTRelements comprising or consisting of a nucleic acid sequence, which isderived from a nucleic acid sequence selected from the group consistingof SEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO:1422 of the patent application WO2013/143700, whose disclosure isincorporated herein by reference, from the homologs of SEQ ID NOs:1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of thepatent application WO2013/143700, from a variant thereof, or preferablyfrom a corresponding RNA sequence. The term “homologs of SEQ ID NOs:1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of thepatent application WO2013/143700” refers to sequences of other speciesthan Homo sapiens, which are homologous to the sequences according toSEQ ID NOs: 1-1363, SEQ ID NO: 1395, SEQ ID NO. 1421 and SEQ ID NO: 1422of the patent application WO2013/143700.

In a preferred embodiment, the 5′-UTR element of the nucleic acidsequence, particularly of the RNA sequence according to the inventioncomprises or consists of a nucleic acid sequence, which is derived froma nucleic acid sequence extending from nucleotide position 5 (i.e. thenucleotide that is located at position 5 in the sequence) to thenucleotide position immediately 5′ to the start codon (located at the3′-end of the sequences), e.g. the nucleotide position immediately 5′ tothe ATG sequence, of a nucleic acid sequence selected from SEQ ID NOs:1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of thepatent application WO2013/143700, from the homologs of SEQ ID NOs:1-1363, SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of thepatent application WO2013/143700 from a variant thereof, or acorresponding RNA sequence. It is particularly preferred that the 5′-UTRelement is derived from a nucleic acid sequence extending from thenucleotide position immediately 3′ to the 5′-TOP to the nucleotideposition immediately 5′ to the start codon (located at the 3′-end of thesequences), e.g. the nucleotide position immediately 5′ to the ATGsequence, of a nucleic acid sequence selected from SEQ ID NOs: 1-1363,SEQ ID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patentapplication WO2013/143700, from the homologs of SEQ ID NOs: 1-1363, SEQID NO: 1395, SEQ ID NO: 1421 and SEQ ID NO: 1422 of the patentapplication WO2013/143700, from a variant thereof, or a correspondingRNA sequence.

In a particularly preferred embodiment, the 5′-UTR element comprises orconsists of a nucleic acid sequence, which is derived from a 5′-UTR of aTOP gene encoding a ribosomal protein or from a variant of a 5′-UTR of aTOP gene encoding a ribosomal protein. For example, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which is derived froma 5′-UTR of a nucleic acid sequence according to any of SEQ ID NOs: 67,170, 193, 244, 259, 554, 650, 675, 700, 721, 913, 1016, 1063, 1120,1138, and 1284-1360 of the patent application WO2013/143700, acorresponding RNA sequence, a homolog thereof, or a variant thereof asdescribed herein, preferably lacking the 5′-TOP motif. As describedabove, the sequence extending from position 5 to the nucleotideimmediately 5′ to the ATG (which is located at the 3′-end of thesequences) corresponds to the 5′-UTR of said sequences.

Preferably, the 5′-UTR element comprises or consists of a nucleic acidsequence, which is derived from a 5″-UTR of a TOP gene encoding aribosomal Large protein (RPL) or from a homolog or variant of a 5′-UTRof a TOP gene encoding a ribosomal Large protein (RPL). For example, the5′-UTR element comprises or consists of a nucleic acid sequence, whichis derived from a 5′-UTR of a nucleic acid sequence according to any ofSEQ ID NOs: 67, 259, 1284-1318, 1344, 1346, 1348-1354, 1357, 1358, 1421and 1422 of the patent application WO2013/143700, a corresponding RNAsequence, a homolog thereof, or a variant thereof as described herein,preferably lacking the 5′-TOP motif.

In a particularly preferred embodiment, the 5′-UTR element comprises orconsists of a nucleic acid sequence which is derived from the 5′-UTR ofa ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, or from a variant of the 5′-UTRof a ribosomal protein Large 32 gene, preferably from a vertebrateribosomal protein Large 32 (L32) gene, more preferably from a mammalianribosomal protein Large 32 (L32) gene, most preferably from a humanribosomal protein Large 32 (L32) gene, wherein preferably the 5′-UTRelement does not comprise the 5′-TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NO: 3529 or 3530(5′-UTR of human ribosomal protein Large 32 lacking the 5′-terminaloligopyrimidine tract: GGCGCTGCCTACGGAGGTGGCAGCCATCTCCTTCTCGGCATC;corresponding to SEQ ID No. 1368 of the patent applicationWO2013/143700) or preferably to a corresponding RNA sequence, or whereinthe at least one 5′-UTR element comprises or consists of a fragment of anucleic acid sequence which has an identity of at least about 40%,preferably of at least about 50%, preferably of at least about 60%,preferably of at least about 70%, more preferably of at least about 80%,more preferably of at least about 90%, even more preferably of at leastabout 95%, even more preferably of at least about 99% to the nucleicacid sequence according to SEQ ID NO: 235 or more preferably to acorresponding RNA sequence (SEQ ID NO: 236), wherein, preferably, thefragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20% etc. of the full-length 5′-UTR.Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In some embodiments, the RNA sequence according to the inventioncomprises a 5′-UTR element, which comprises or consists of a nucleicacid sequence, which is derived from the 5′-UTR of a vertebrate TOPgene, such as a mammalian, e.g. a human TOP gene, selected from RPSA,RPS2, RPS3, RPS3A, RPS4, RPS5, RPS6, RPS7, RPS8, RPS9, RPS10, RPS11,RPS12, RPS13, RPS14, RPS15, RPS15A, RPS16, RPS17, RPS18, RPS19, RPS20,RPS21, RPS23, RPS24, RPS25, RPS26, RPS27, RPS27A, RPS28, RPS29, RPS30,RPL3, RPL4, RPL5, RPL6, RPL7, RPL7A, RPL8, RPL9, RPL10, RPL10A, RPL11,RPL12, RPL13, RPL13A, RPL14, RPL15, RPL17, RPL18, RPL18A, RPL19, RPL21,RPL22, RPL23, RPL23A, RPL24, RPL26, RPL27, RPL27A, RPL28, RPL29, RPL30,RPL31, RPL32, RPL34, RPL35, RPL35A, RPL36, RPL36A, RPL37, RPL37A, RPL38,RPL39, RPL40, RPL41, RPLP0, RPLP1, RPLP2, RPLP3, RPLP0, RPLP1, RPLP2,EEF1A1, EEF1B2, EEF1D, EEF1G, EEF2, EIF3E, EIF3F, EIF3H, EIF2S3, EIF3C,EIF3K, EIF3EIP, EIF4A2, PABPC1, HNRNPA1, TPT1, TUBB1, UBA52, NPM1,ATP5G2, GNB2L1, NME2, UQCRB, or from a homolog or variant thereof,wherein preferably the 5′-UTR element does not comprise a TOP-motif orthe 5′-TOP of said genes, and wherein optionally the 5′-UTR elementstarts at its 5′-end with a nucleotide located at position 1, 2, 3, 4,5, 6, 7, 8, 9 or 10 downstream of the 5′-terminal oligopyrimidine tract(TOP) and wherein further optionally the 5′-UTR element which is derivedfrom a 5′-UTR of a TOP gene terminates at its 3′-end with a nucleotidelocated at position 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 upstream of thestart codon (A(U/T)G) of the gene it is derived from.

In further particularly preferred embodiments, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which is derived fromthe 5′-UTR of a ribosomal protein Large 32 gene (RPL32), a ribosomalprotein Large 35 gene (RPL35), a ribosomal protein Large 21 gene(RPL21), an ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, an hydroxysteroid(17-beta) dehydrogenase 4 gene (HSD17B4), an androgen-induced 1 gene(AIG1), cytochrome c oxidase subunit Vic gene (COX6C), or aN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, preferably from a vertebrate ribosomal proteinLarge 32 gene (RPL32), a vertebrate ribosomal protein Large 35 gene(RPL35), a vertebrate ribosomal protein Large 21 gene (RPL21), avertebrate ATP synthase, H+ transporting, mitochondrial F1 complex,alpha subunit 1, cardiac muscle (ATP5A1) gene, a vertebratehydroxysteroid (17-beta) dehydrogenase 4 gene (HSD17B4), a vertebrateandrogen-induced 1 gene (AIG1), a vertebrate cytochrome c oxidasesubunit Vic gene (COX6C), or a vertebrate N-acylsphingosineamidohydrolase (acid ceramidase) 1 gene (ASAH1) or from a variantthereof, more preferably from a mammalian ribosomal protein Large 32gene (RPL32), a ribosomal protein Large 35 gene (RPL35), a ribosomalprotein Large 21 gene (RPL21), a mammalian ATP synthase, H+transporting, mitochondrial F1 complex, alpha subunit 1, cardiac muscle(ATP5A1) gene, a mammalian hydroxysteroid (17-beta) dehydrogenase 4 gene(HSD17B4), a mammalian androgen-induced 1 gene (AIG1), a mammaliancyto-chrome c oxidase subunit Vic gene (COX6C), or a mammalianN-acylsphingosine ami-dohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, most preferably from a human ribosomal proteinLarge 32 gene (RPL32), a human ribosomal protein Large 35 gene (RPL35),a human ribosomal protein Large 21 gene (RPL21), a human ATP synthase,H+ transporting, mitochondrial F1 complex, alpha subunit 1, cardiacmuscle (ATP5A1) gene, a human hydroxysteroid (17-beta) dehydrogenase 4gene (HSD17B4), a human androgen-induced 1 gene (AIG1), a humancytochrome c oxidase subunit Vic gene (COX6C), or a humanN-acylsphingosine amidohydrolase (acid ceramidase) 1 gene (ASAH1) orfrom a variant thereof, wherein preferably the 5′-UTR element does notcomprise the 5′TOP of said gene.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NOs: 1412-1420 ofthe patent application WO2013/143700, or a corresponding RNA sequence orwherein the at least one 5′-UTR element comprises or consists of afragment of a nucleic acid sequence which has an identity of at leastabout 40%, preferably of at least about 50%, preferably of at leastabout 60%, preferably of at least about 70%, more preferably of at leastabout 80%, more preferably of at least about 90%, even more preferablyof at least about 95%, even more preferably of at least about 99% to thenucleic acid sequence according SEQ ID NOs: 1412-1420 of the patentapplication WO2013/143700, wherein, preferably, the fragment is asdescribed above, i.e. being a continuous stretch of nucleotidesrepresenting at least 20% etc. of the full-length 5′-UTR. Preferably,the fragment exhibits a length of at least about 20 nucleotides or more,preferably of at least about 30 nucleotides or more, more preferably ofat least about 40 nucleotides or more. Preferably, the fragment is afunctional fragment as described herein.

Accordingly, in a particularly preferred embodiment, the 5′-UTR elementcomprises or consists of a nucleic acid sequence, which has an identityof at least about 40%, preferably of at least about 50%, preferably ofat least about 60%, preferably of at least about 70%, more preferably ofat least about 80%, more preferably of at least about 90%, even morepreferably of at least about 95%, even more preferably of at least about99% to the nucleic acid sequence according to SEQ ID NOs: 237 or 238(5′-UTR of ATP5A1 lacking the 5′-terminal oligopyrimidine tract:GCGGCTCGGCCATTTTGTCCCAGTCAGTCCGGAGGCTGCGGCTGCAGAAGTACCGCCTGCGGAGTAACTGCAAAG; corresponding to SEQ ID NO: 1414 of the patent applicationWO2013/143700) or preferably to a corresponding RNA sequence, or whereinthe at least one 5′-UTR element comprises or consists of a fragment of anucleic acid sequence which has an identity of at least about 40%,preferably of at least about 50%, preferably of at least about 60%,preferably of at least about 70%, more preferably of at least about 80%,more preferably of at least about 90%, even more preferably of at leastabout 95%, even more preferably of at least about 99% to the nucleicacid sequence according to SEQ ID NO: 237 or more preferably to acorresponding RNA sequence (SEQ ID NO: 238), wherein, preferably, thefragment is as described above, i.e. being a continuous stretch ofnucleotides representing at least 20% etc. of the full-length 5′-UTR.Preferably, the fragment exhibits a length of at least about 20nucleotides or more, preferably of at least about 30 nucleotides ormore, more preferably of at least about 40 nucleotides or more.Preferably, the fragment is a functional fragment as described herein.

In embodiments, the artificial nucleic acid as defined herein,particularly the RNA as defined herein comprises a 5′-UTR element, whichmay be any 5′-UTR element described in WO2016/107877. In this context,the disclosure of WO2016/107877 relating to 5′-UTR elements/sequences isherewith incorporated by reference. Particularly preferred 5′-UTRelements are nucleic acid sequences according to SEQ ID NOs: 25 to 30and SEQ ID NOs: 319 to 382 of the patent application WO2016/107877, orfragments or variants of these sequences. In this context, it isparticularly preferred that the 5′-UTR element of the RNA sequenceaccording to the present invention comprises or consists of acorresponding RNA sequence of the nucleic acid sequence according SEQ IDNOs: 25 to 30 and SEQ ID NOs: 319 to 382 of the patent applicationWO2016/107877.

In embodiments, the artificial nucleic acid sequence as defined herein,particularly the RNA as defined herein comprises a 5′-UTR element, whichmay be any 5′-UTR element as described in WO2017/036580. In thiscontext, the disclosure of WO2017/036580 relating to 5′-UTRelements/sequences is herewith incorporated by reference. Particularlypreferred 5′-UTR elements are nucleic acid sequences according to SEQ IDNOs: 1 to 151 of the patent application WO2017/036580, or fragments orvariants of these sequences. In this context, it is particularlypreferred that the 5′-UTR element of the RNA sequence according to thepresent invention comprises or consists of a corresponding RNA sequenceof the nucleic acid sequence according to SEQ ID NOs: 1 to 151 of thepatent application WO2017/036580.

Preferably, the at least one 5′-UTR element and the at least one 3′-UTRelement act synergistically to increase protein production from the atleast one RNA sequence as described above.

According to a preferred embodiment the RNA sequence according to theinvention comprises, preferably in 5″- to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b.) a 5′-UTR element which comprises or consists of a nucleic acid    sequence which is derived from the 5′-UTR of a TOP gene (SEQ ID NOs:    235-238), a homolog, a fragment or a variant thereof;-   c1.) optionally, at least one coding sequence encoding at least one    heterologous secretory signal sequence;-   c2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Henipavirus protein or peptide as    defined herein or a fragment or variant thereof.-   d.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from a gene providing a stable RNA,    preferably comprising or consisting of the corresponding RNA    sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or    252, a homolog, a fragment or a variant thereof;-   e.) optionally, a poly(A) sequence preferably comprising 64    adenosines; and-   f.) optionally, a poly(C) sequence, preferably comprising 30    cytosines.

According to a particularly preferred embodiment the RNA sequenceaccording to the invention comprises, preferably in 5′- to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b.) a 5′-UTR element which comprises or consists of a nucleic acid    sequence which is derived from the 5′-UTR of a TOP gene (SEQ ID NOs:    235-238), a homolog, a fragment or a variant thereof;-   c1.) optionally, at least one coding sequence encoding at least one    heterologous secretory signal sequence, preferably selected from SEQ    ID NOs 317-572;-   c2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Hendra virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid sequences according to SEQ ID NOs:    34-37, 45-52, 60-63, 71-78, 86-89, 97-104, 112-115, 123-130,    138-141, 149-156, 164-167, 175-182, 190-193, 201-208, 216-219,    227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739,    762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728,    747-754, 773-780, 799-806, 840-843, 866-869, 892-895, 918-921,    944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884, 903-910,    929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077,    1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233,    1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196,    1215-1222, 1241-1248, 1267-1274, or a fragment or variant thereof;-   d.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from a gene providing a stable RNA,    preferably comprising or consisting of the corresponding RNA    sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or    252, a homolog, a fragment or a variant thereof;-   e.) optionally, a poly(A) sequence preferably comprising 64    adenosines; and-   f.) optionally, a poly(C) sequence, preferably comprising 30    cytosines.

According to a particularly preferred embodiment the RNA sequenceaccording to the invention comprises, preferably in 5′- to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b.) a 5′-UTR element which comprises or consists of a nucleic acid    sequence which is derived from the 5′-UTR of a TOP gene (SEQ ID NOs:    235-238), a homolog, a fragment or a variant thereof;-   c1.) optionally, at least one coding sequence encoding at least one    heterologous secretory signal sequence, preferably selected from SEQ    ID NOs 317-572;-   c2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Nipah virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid sequences according to SEQ ID NOs:    27-33, 38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137,    142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226,    599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761,    781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,    766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943,    963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928,    948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099,    1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,    1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214,    1234-1240, 1260-1266, 1516-1539 or a fragment or variant thereof;-   d.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from a gene providing a stable RNA,    preferably comprising or consisting of the corresponding RNA    sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or    252, a homolog, a fragment or a variant thereof;-   e.) optionally, a poly(A) sequence preferably comprising 64    adenosines; and-   f.) optionally, a poly(C) sequence, preferably comprising 30    cytosines.

Histone Stem-Loop:

In a particularly preferred embodiment, the nucleic acid sequence,particularity the RNA sequence according to the invention comprises ahistone stem-loop sequence/structure. Such histone stem-loop sequencesare preferably selected from histone stem-loop sequences as disclosed inWO 2012/019780, the disclosure of which is incorporated herewith byreference.

A histone stem-loop sequence suitable to be used within the presentinvention is preferably derived from formulae (I) or (II) of the patentapplication WO2012/019780, herewith incorporated by reference. Accordingto a further preferred embodiment the RNA as defined herein may compriseat least one histone stem-loop sequence derived from at least one of thespecific formulae (Ia) or (IIa) of the patent application WO2012/019780.

A particular preferred histone stem-loop sequence is the sequenceaccording to SEQ ID NO: 253 or more preferably the corresponding RNAsequence according to SEQ ID NO: 254.

Accordingly, in preferred embodiments, the artificial nucleic acid ofthe invention comprises at least one histone stem-loop as definedherein. Preferably, the at least one histone stem loop comprises anucleic acid sequence according to SEQ ID NOs: 253 or 254, or a fragmentor variant thereof.

In particularly preferred embodiments, the artificial nucleic acid,preferably the artificial mRNA of the invention comprises a 3′-terminalsequence element comprising a poyl(A)sequence as defined herein and ahistons-stem-loop sequence as defined herein, wherein the 3′-terminalsequence element may be selected from SEQ ID NOs: 1509, 1510, 1511 or1512.

mRNA Structures:

Any of the above modifications may be applied to the nucleic acidsequence, in particular, to the DNA and/or RNA sequence of the presentinvention, and further to any DNA or RNA as used in the context of thepresent invention and may be, if suitable or necessary, be combined witheach other in any combination, provided, these combinations ofmodifications do not interfere with each other in the respective nucleicacid sequence. A person skilled in the art will be able to take hischoice accordingly.

The artificial nucleic acid sequence according to the invention,particularly the RNA sequence according to the present invention whichcomprises at least one coding sequence as defined herein, may preferablycomprise a 5′-UTR and/or a 3′-UTR preferably containing at least onehistone stem-loop. The 3′-UTR of the RNA sequence according to theinvention preferably comprises also a poly(A) and/or a poly(C) sequenceas defined herein. The single elements of the 3′-UTR may occur thereinin any order from 5′ to 3′ along the sequence of the RNA sequence of thepresent invention. In addition, further elements as described herein,may also be contained, such as a stabilizing sequence as defined herein(e.g. derived from the UTR of a globin gene), IRES sequences, etc. Eachof the elements may also be repeated in the RNA sequence according tothe invention at least once (particularly in di- or multicistronicconstructs), preferably twice or more. As an example, the singleelements may be present in the nucleic acid sequence, particularly inthe RNA sequence according to the invention in the following order:

5′-coding sequence-histone stem-loop-poly(A)/(C) sequence-3′; or

5′-coding sequence-poly(A)/(C) sequence-histone stem-loop-3′; or

5′-coding sequence-histone stem-loop-polyadenylation signal-3′; or

5′-coding sequence-polyadenylation signal-histone stem-loop-3′; or

5′-coding sequence-histone stem-loop-histone stem-loop-poly(A)/(C)sequence-3′; or

5′-coding sequence-histone stem-loop-histone stem-loop-polyadenylationsignal-3′; or

5′-coding sequence-stabilizing sequence-poly(A)/(C) sequence-histonestem-loop-3′; or 5′-coding sequence-stabilizing sequence-poly(A)/(C)sequence-poly(A)/(C) sequence-histone stem-loop-3″; etc.

According to a further embodiment, the nucleic acid sequence of thepresent invention, particularly the RNA sequence of the presentinvention, preferably comprises at least one of the following structuralelements: a 5′- and/or 3′-untranslated region element (UTR element),particularly a 5′-UTR element, which preferably comprises or consists ofa nucleic acid sequence which is derived from the 5′-UTR of a TOP geneor from a fragment, homolog or a variant thereof, or a 5′- and/or 3′-UTRelement which may preferably be derivable from a gene that provides astable RNA or from a homolog, fragment or variant thereof; ahistone-stem-loop structure, preferably a histone-stem-loop in its 3′untranslated region; a 5′-cap structure; a poly-A tail; or a poly(C)sequence.

In preferred embodiments the nucleic acid sequence, in particular, theRNA sequence comprises, preferably in 5′- to 3′-direction, the followingelements a)-h):

-   -   a) 5′-cap structure, preferably as defined herein;    -   b) optionally, 5′-UTR element, preferably as defined herein;    -   c) at least one coding sequence, preferably as defined herein;    -   d) a 3′-UTR element, preferably as defined herein;    -   e) optionally, poly(A) sequence, preferably as defined herein;    -   f) optionally, poly(C) sequence, preferably as defined herein;    -   g) optionally, a histone stem-loop, preferably as defined        herein; and    -   h) optionally, a 3′-terminal sequence element as defined herein.

In a preferred embodiment the nucleic acid sequence, in particular, theRNA sequence comprises, preferably in 5′- to 3′-direction:

-   a) a 5′-cap structure, preferably m7GpppN;-   b1.) optionally, at least one coding sequence encoding at least one    heterologuous secretory signal sequence;-   b2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Henipavirus protein or peptide as    defined herein or a fragment or variant thereof;-   c.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from an alpha globin gene, preferably    comprising the corresponding RNA sequence of the nucleic acid    sequence according to SEQ ID NO: 246, a homolog, a fragment or a    variant thereof;-   d.) optionally, a poly(A) sequence, preferably comprising 64    adenosines;-   e.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;-   f.) optionally, a histone stem-loop, preferably comprising the RNA    sequence according to SEQ ID NO: 254;-   g.) optionally, a poly(A) sequence and a histone stem-loop    comprising the RNA sequence according to SEQ ID NOs: 1509, 1510,    1511 or 1512

In a particularly preferred embodiment the nucleic acid sequence, inparticular, the RNA sequence comprises, preferably in 5′- to3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b1.) optionally, at least one coding sequence encoding at least one    heterologuous secretory signal sequence according to SEQ ID NOs:    317-572;-   b2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Hendra virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid according to 34-37, 45-52, 60-63,    71-78, 86-89, 97-104, 112-115, 123-130, 138-141, 149-156, 164-167,    175-182, 190-193, 201-208, 216-219, 227-234, 606-609, 632-635,    658-661, 684-687, 710-713, 736-739, 762-765, 788-791, 617-624,    643-650, 669-676, 695-702, 721-728, 747-754, 773-780, 799-806,    840-843, 866-869, 892-895, 918-921, 944-947, 970¬-973, 996-999,    1022-1025, 851-858, 877-884, 903-910, 929-936, 955-962, 981-988,    1007-1014, 1033-1040, 1074-1077, 1100-1103, 1126-1129, 1152-1155,    1178-1181, 1204-1207, 1230-1233, 1256-1259, 1085-1092, 1111-1118,    1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248, 1267-1274 or    a fragment or variant thereof;-   c.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from an alpha globin gene, preferably    comprising the corresponding RNA sequence of the nucleic acid    sequence according to SEQ ID NO: 246, a homolog, a fragment or a    variant thereof;-   d.) optionally, a poly(A) sequence, preferably comprising 64    adenosines;-   e.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;-   f.) optionally, a histone stem-loop, preferably comprising the RNA    sequence according to SEQ ID NO: 254;-   g.) optionally, a poly(A) sequence and a histone stem-loop    comprising the RNA sequence according to SEQ ID NOs: 1509, 1510,    1511 or 1512

In a further particularly preferred embodiment the nucleic acidsequence, in particular, the RNA sequence comprises, preferably in 5′-to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b1.) optionally, at least one coding sequence encoding at least one    heterologuous secretory signal sequence according to SEQ ID NOs:    317-572;-   b2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Nipah virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid according to SEQ ID NOs: 27-33,    38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137,    142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226,    599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761,    781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,    766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943,    963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928,    948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099,    1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,    1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214,    1234-1240, 1260-1266, 1516-1539 or a fragment or variant thereof;-   c.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from an alpha globin gene, preferably    comprising the corresponding RNA sequence of the nucleic acid    sequence according to SEQ ID NO: 246, a homolog, a fragment or a    variant thereof;-   d.) optionally, a poly(A) sequence, preferably comprising 64    adenosines;-   e.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;-   f.) optionally, a histone stem-loop, preferably comprising the RNA    sequence according to SEQ ID NO: 254;-   g.) optionally, a poly(A) sequence and a histone stem-loop    comprising the RNA sequence according to SEQ ID NOs: 1509, 1510,    1511 or 1512

According to another particularly preferred embodiment the nucleic acidsequence, in particular, the RNA sequence according to the inventioncomprises, preferably in 5′- to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b.) a 5′-UTR element which comprises or consists of a nucleic acid    sequence which is derived from the 5′-UTR of a TOP gene (SEQ ID NOs:    235-238), a homolog, a fragment or a variant thereof;-   c1.) optionally, at least one coding sequence encoding at least one    heterologuous secretory signal sequence according to SEQ ID NOs:    317-572;-   c2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Hendra virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid sequences according to SEQ ID NOs:    34-37, 45-52, 60-63, 71-78, 86-89, 97-104, 112-115, 123-130,    138-141, 149-156, 164-167, 175-182, 190-193, 201-208, 216-219,    227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739,    762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728,    747-754, 773-780, 799-806, 840-843, 866-869, 892-895, 918-921,    944-947, 970¬-973, 996-999, 1022-1025, 851-858, 877-884, 903-910,    929-936, 955-962, 981-988, 1007-1014, 1033-1040, 1074-1077,    1100-1103, 1126-1129, 1152-1155, 1178-1181, 1204-1207, 1230-1233,    1256-1259, 1085-1092, 1111-1118, 1137-1144, 1163-1170, 1189-1196,    1215-1222, 1241-1248, 1267-1274 or a fragment or variant thereof;-   d.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from a gene providing a stable RNA,    preferably comprising or consisting of the corresponding RNA    sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or    252, a homolog, a fragment or a variant thereof;-   e.) optionally, a poly(A) sequence preferably comprising 64    adenosines;-   f.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;-   g.) optionally, a histone stem-loop, preferably comprising the RNA    sequence according to SEQ ID NO: 254;-   h.) optionally, a poly(A) sequence and a histone stem-loop    comprising the RNA sequence according to SEQ ID NOs: 1509, 1510,    1511 or 1512.

According to another particularly preferred embodiment the nucleic acidsequence, in particular, the RNA sequence according to the inventioncomprises, preferably in 5′- to 3′-direction:

-   a.) a 5′-cap structure, preferably m7GpppN;-   b.) a 5′-UTR element which comprises or consists of a nucleic acid    sequence which is derived from the 5′-UTR of a TOP gene (SEQ ID NOs:    235-238), a homolog, a fragment or a variant thereof;-   c1.) optionally, at least one coding sequence encoding at least one    heterologuous secretory signal sequence according to SEQ ID NOs:    317-572;-   c2.) at least one coding sequence encoding at least one antigenic    peptide or protein derived from a Nipah virus protein or peptide or    a fragment or variant thereof, preferably comprising or consisting    of any one of the nucleic acid sequences according to SEQ ID NOs:    27-33, 38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122, 131-137,    142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226,    599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761,    781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,    766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943,    963-969, 989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928,    948-954, 974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099,    1119-1125, 1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255,    1078-1084, 1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214,    1234-1240, 1260-1266, 1516-1539 or a fragment or variant thereof;-   d.) a 3′-UTR element comprising or consisting of a nucleic acid    sequence which is derived from a gene providing a stable RNA,    preferably comprising or consisting of the corresponding RNA    sequence of a nucleic acid sequence according to SEQ ID NOs: 250 or    252, a homolog, a fragment or a variant thereof;-   e.) optionally, a poly(A) sequence preferably comprising 64    adenosines;-   f.) optionally, a poly(C) sequence, preferably comprising 30    cytosines;-   g.) optionally, a histone stem-loop, preferably comprising the RNA    sequence according to SEQ ID NO: 254;-   h.) optionally, a poly(A) sequence and a histone stem-loop    comprising the RNA sequence according to SEQ ID NOs: 1509, 1510,    1511 or 1512.

Preferred Hendra and Nipah Constructs of the Invention:

In the following, preferred and particularly suitable Hendra virus andNipah virus mRNA sequences of the invention are provided.

Preferred Hendra polypeptide, nucleic acid and mRNA sequences areprovided in Table 5. Therein, each row (row 1-36) represents a specificsuitable Hendra virus construct of the invention. The proteindesign/name is indicated for each row (column “Name”). Accession numbersare provided in the <223> identifier of the respective SEQ ID NOs in thesequence listing. Column “SEQ ID NO: Protein” provides the respectiveSEQ ID NOs of the protein constructs as provided in the sequencelisting. mRNA constructs comprising coding sequences encoding saidproteins are provided in column “SEQ ID NO: mRNA design 1” column “SEQID NO: mRNA design 2” and column “SEQ ID NO: mRNA design 3”. Additionalinformation regarding each of the sequences provided in Table 5 may alsobe derived from the sequence listing, in particular from the detailsprovided therein under identifier <223>.

TABLE 5 Preferred Hendra virus polypeptide, nucleic acid and mRNAsequences SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: Row Name ProteinmRNA design 1 mRNA design 2 mRNA design 3 1 F 8 1282 1360 1438 2 F 91283 1361 1439 3 F 10 1284 1362 1440 4 F 11 1285 1363 1441 5HsIgE(1-18)_F(27-546) 814 1308 1386 1464 6 HsIgE(1-18)_F(27-546) 8151309 1387 1465 7 HsIgE(1-18)_F(26-546) 816 1310 1388 1466 8HsIgE(1-18)_F(27-546) 817 1311 1389 1467 9 H1N1-HA(1-17)_F(27-546) 10481334 1412 1490 10 H1N1-HA(1-17)_F(27-546) 1049 1335 1413 1491 11H1N1-HA(1-17)_F(26-546) 1050 1336 1414 1492 12 H1N1-HA(1-17)_F(27-546)1051 1337 1415 1493 13 G 19 1293 1371 1449 14 G 20 1294 1372 1450 15 G21 1295 1373 1451 16 G 22 1296 1374 1452 17 G 23 1297 1375 1453 18 G 241298 1376 1454 19 G 25 1299 1377 1455 20 G 26 1300 1378 1456 21HsIgE(1-18)_G(70-604) 825 1319 1397 1475 22 HsIgE(1-18)_G(70-604) 8261320 1398 1476 23 HsIgE(1-18)_G(70-604) 827 1321 1399 1477 24HsIgE(1-18)_G(70-604) 828 1322 1400 1478 25 HsIgE(1-18)_G(70-604) 8291323 1401 1479 26 HsIgE(1-18)_G(70-604) 830 1324 1402 1480 27HsIgE(1-18)_G(70-604) 831 1325 1403 1481 28 HsIgE(1-18)_G(70-604) 8321326 1404 1482 29 H1N1-HA(1-17)_G(70-604) 1059 1345 1423 1501 30H1N1-HA(1-17)_G(70-604) 1060 1346 1424 1502 31 H1N1-HA(1-17)_G(70-604)1061 1347 1425 1503 32 H1N1-HA(1-17)_G(70-604) 1062 1348 1426 1504 33H1N1-HA(1-17)_G(70-604) 1063 1349 1427 1505 34 H1N1-HA(1-17)_G(70-604)1064 1350 1428 1506 35 H1N1-HA(1-17)_G(70-604) 1065 1351 1429 1507 36H1N1-HA(1-17)_G(70-604) 1066 1352 1430 1508

Preferred Nipah polypeptide, nucleic acid and mRNA sequences areprovided in Table 6. Therein, each row (row 1-42) represents a specificsuitable Nipah virus construct of the invention. The protein design/nameis indicated for each row (column “Name”). Accession numbers areprovided in the <223> identifier of the respective SEQ ID NOs in thesequence listing. Column “SEQ ID NO: Protein” provides the respectiveSEQ ID NOs of the protein constructs as provided in the sequencelisting. mRNA constructs comprising coding sequences encoding saidproteins are provided in column “SEQ ID NO: mRNA design 1” column “SEQID NO: mRNA design 2” and column “SEQ ID NO: mRNA design 3”. Additionalinformation regarding each of the sequences provided in Table 5 may alsobe derived from the sequence listing, in particular from the detailsprovided therein under identifier <223>.

TABLE 6 Preferred Nipah virus polypeptide, nucleic acid and mRNAsequences SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID NO: Row Name ProteinmRNA design 1 mRNA design 2 mRNA design 3 1 F 1 1275 1353 1431 2 F 21276 1354 1432 3 F 3 1277 1355 1433 4 F 4 1278 1356 1434 5 F 5 1279 13571435 6 F 6 1280 1358 1436 7 F 7 1281 1359 1437 8 HsIgE(1-18)_F(27-546)807 1301 1379 1457 9 HsIgE(1-18)_F(27-546) 808 1302 1380 1458 10HsIgE(1-18)_F(27-546) 809 1303 1381 1459 11 HsIgE(1-18)_F(27-546) 8101304 1382 1460 12 HsIgE(1-18)_F(27-546) 811 1305 1383 1461 13HsIgE(1-18)_F(27-546) 812 1306 1384 1462 14 HsIgE(1-18)_F(27-546) 8131307 1385 1463 15 H1N1-HA(1-17)_F(27-546) 1041 1327 1405 1483 16H1N1-HA(1-17)_F(27-546) 1042 1328 1406 1484 17 H1N1-HA(1-17)_F(27-546)1043 1329 1407 1485 18 H1N1-HA(1-17)_F(27-546) 1044 1330 1408 1486 19H1N1-HA(1-17)_F(27-546) 1045 1331 1409 1487 20 H1N1-HA(1-17)_F(27-546)1046 1332 1410 1488 21 H1N1-HA(1-17)_F(27-546) 1047 1333 1411 1489 22 G12 1286 1364 1442 23 G 13 1287 1365 1443 24 G 14 1288 1366 1444 25 G 151289 1367 1445 26 G 16 1290 1368 1446 27 G 17 1291 1369 1447 28 G 181292 1370 1448 29 HsIgE(1-18)_G(70-602) 818 1312 1390 1468 30HsIgE(1-18)_G(70-602) 819 1313 1391 1469 31 HsIgE(1-18)_G(70-602) 8201314 1392 1470 32 HsIgE(1-18)_G(70-602) 821 1315 1393 1471 33HsIgE(1-18)_G(70-602) 822 1316 1394 1472 34 HsIgE(1-18)_G(70-602) 8231317 1395 1473 35 HsIgE(1-18)_G(70-602) 824 1318 1396 1474 36H1N1-HA(1-17)_G(70-602) 1052 1338 1416 1494 37 H1N1-HA(1-17)_G(70-602)1053 1339 1417 1495 38 H1N1-HA(1-17)_G(70-602) 1054 1340 1418 1496 39H1N1-HA(1-17)_G(70-602) 1055 1341 1419 1497 40 H1N1-HA(1-17)_G(70-602)1056 1342 1420 1498 41 H1N1-HA(1-17)_G(70-602) 1057 1343 1421 1499 42H1N1-HA(1-17)_G(70-602) 1058 1344 1422 1500 43 HsSPARC(1-17)_F(27-546)1513 1540 1543 1546 44 HsCTRB2(1-18)_F(27-546) 1514 1541 1544 1547 45Nipah 1515 1542 1545 1548 henipavirus_AAK50553_F(1- 26)F(27-546)

Accordingly, it is particularly preferred that the nucleic acid sequenceaccording to the invention comprises or consists of a nucleic acidsequence selected from sequences being identical or at least 50%, 60%,70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the mRNA sequences according to SEQ IDNOs: 1275-1508, 1540-1548 or a fragment or variant thereof.

Accordingly, it is particularly preferred that the nucleic acid sequenceaccording to the invention comprises or consists of a nucleic acidsequence selected from sequences being identical or at least 50%, 60%,70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the mRNA sequences according to SEQ IDNOs: 1282-1285, 1293-1300, 1308-1311, 1319-1326, 1334-1337, 1345-1352,1360-1363, 1371-1378, 1386-1389, 1397-1404, 1412-1415, 1423-1430,1438-1441, 1464-1467, 1490-1493, 1449-1456, 1475-1482, 1501-1508 or afragment or variant thereof.

Accordingly, it is particularly preferred that the nucleic acid sequenceaccording to the invention comprises or consists of a nucleic acidsequence selected from sequences being identical or at least 50%, 60%,70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, or 99% identical to the mRNA sequences according to SEQ IDNOs: 1275-1281, 1286-1292, 1301-1307, 1312-1318, 1327-1333, 1338-1344,1353-1359, 1364-1370, 1379-1385, 1390-1396, 1405-1411, 1416-1422,1431-1437, 1457-1463, 1483-1489, 1442-1448, 1468-1474, 1494-1500,1540-1548 or a fragment or variant thereof.

Composition:

In a further aspect, the present invention concerns a compositioncomprising at least one artificial nucleic acid comprising at least onecoding sequence as defined herein and a pharmaceutically acceptablecarrier. The composition according to the invention is preferablyprovided as a pharmaceutical composition or as a vaccine.

According to a preferred embodiment, the (pharmaceutical) composition orthe vaccine according to the invention comprises at least one nucleicacid of the present invention, wherein the at least one coding sequenceof the at least one nucleic acid sequence encodes at least oneHenipavirus peptide or protein selected from Henipavirus RNA-directedRNA polymerase (L), Henipavirus fusion protein (F), Henipavirusnon-structural protein (V), Henipavirus glycoprotein (G), Henipavirusnucleoprotein (N), Henipavirus matrix protein (M), Henipavirusphosphoprotein (P), Henipavirus protein C, and Henipavirus protein W, aswell as to fragments or variants of all these proteins.

The (pharmaceutical) composition or vaccine according to the inventionmay thus comprise at least one nucleic acid comprising at least onenucleic acid sequence comprising at least one coding region, encoding atleast one Henipavirus antigenic peptide or protein, particularly, atleast one Henipavirus protein selected from Henipavirus RNA-directed RNApolymerase (L), Henipavirus fusion protein (F), Henipavirusnon-structural protein (V), Henipavirus glycoprotein (G), Henipavirusnucleoprotein (N), Henipavirus matrix protein (M), Henipavirusphosphoprotein (P), Henipavirus protein C, and Henipavirus protein W, afragment or variant thereof, wherein the at least one coding region ofthe at least one nucleic acid sequence encodes one specific Henipavirusantigenic peptide or protein as defined herein or a fragment or avariant thereof.

According to a further preferred embodiment, the (pharmaceutical)composition or the vaccine according to the invention comprises at leastone nucleic acid of the present invention, wherein the at least onecoding sequence of the at least one nucleic acid sequence encodes atleast one Hendra virus peptide or protein selected from Hendra virusRNA-directed RNA polymerase (L), Hendra virus fusion protein (F), Hendravirus non-structural protein (V), Hendra virus glycoprotein (G), Hendravirus nucleoprotein (N), Hendra virus matrix protein (M), Hendra virusphosphoprotein (P), Hendra virus protein C, and Hendra virus protein W,as well as to fragments or variants of all these proteins.

In a particularly preferred embodiment, the (pharmaceutical) compositionor the vaccine according to the invention comprises at least one nucleicacid of the present invention, wherein the at least one coding sequenceof the at least one nucleic acid sequence encodes at least one Hendravirus peptide or protein selected from Hendra virus fusion protein (F)and Hendra virus glycoprotein (G) (and optionally a secretory signalsequence) as well as to fragments or variants of all these proteins,preferably proteins or peptides according to SEQ ID NOs: 8-11, 19-26,580-583, 591-598, 814-817, 825-832, 1048-1051, 1059-1066 or a homolog,fragment or variant of any of these sequences (see Table 1 and Table 1B,column “A”).

Preferably, the (pharmaceutical) composition or the vaccine according tothe invention comprises at least one nucleic acid of the presentinvention, wherein the at least one coding sequence of the at least onenucleic acid sequence encodes at least one Hendra virus peptide orprotein selected from Hendra virus fusion protein (F) and Hendra virusglycoprotein (G) (and, optionally, a secretory signal sequence) as wellas to fragments or variants of all these proteins, wherein the Hendravirus peptide or protein preferably comprises or consists of an aminoacid sequence having a sequence identity of at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, morepreferably of at least 80%, even more preferably at least 85%, even morepreferably of at least 90% and most preferably of at least 95% or even97%, with any one of the amino acid sequences according to SEQ ID NOs:8-11, 19-26, 580-583, 591-598, 814-817, 825-832, 1048-1051, 1059-1066,or a homolog, fragment or variant of any of these sequences (see Table 1and Table 1B, column “A”) fragment or variant of any one of thesesequences.

More preferably, the (pharmaceutical) composition or the vaccineaccording to the invention comprises at least one nucleic acidcomprising at least one nucleic acid sequence of the present invention,wherein the at least one coding sequence of the at least one nucleicacid sequence comprises or consists of a nucleic acid sequence encodingat least one Hendra virus peptide or protein (and, optionally, asecretory signal sequence) preferably comprises or consists of an aminoacid sequence having a sequence identity of at least 80% with any one ofthe amino acid sequences according to SEQ ID NOs: 8-11, 19-26, 580-583,591-598, 814-817, 825-832, 1048-1051, 1059-1066, or a homolog, fragmentor variant of any of these sequences (see Table 1 and Table 1B, column“A”) fragment or variant of any one of these sequences.

In preferred embodiments, the (pharmaceutical) composition or thevaccine according to the invention comprises at least one nucleic acidcomprising at least one nucleic acid sequence of the present invention,wherein the at least one coding sequence (encoding at least one Hendravirus antigenic peptide or protein, and, optionally, a secretory signalsequence) of the at least one nucleic acid sequence comprises orconsists of any one of the nucleic acid sequences according to SEQ IDNOs: 34-37, 45-52, 60-63, 71-78, 86-89, 97-104, 112-115, 123-130,138-141, 149-156, 164-167, 175-182, 190-193, 201-208, 216-219, 227-234,606-609, 632-635, 658-661, 684-687, 710-713, 736-739, 762-765, 788-791,617-624, 643-650, 669-676, 695-702, 721-728, 747-754, 773-780, 799-806,840-843, 866-869, 892-895, 918-921, 944-947, 970¬-973, 996-999,1022-1025, 851-858, 877-884, 903-910, 929-936, 955-962, 981-988,1007-1014, 1033-1040, 1074-1077, 1100-1103, 1126-1129, 1152-1155,1178-1181, 1204-1207, 1230-1233, 1256-1259, 1085-1092, 1111-1118,1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248, 1267-1274 (asdefined in Table 1 and Table 1B) or a fragment or variant of any one ofthese sequences.

According to another embodiment, the (pharmaceutical) composition or thevaccine according to the invention comprises at least one nucleic acidcomprising at least one nucleic acid sequence of the present invention,wherein the at least one coding sequence (encoding at least one Hendravirus antigenic peptide or protein, and, optionally, a secretory signalsequence) of the at least one nucleic acid sequence comprises orconsists of a nucleic acid sequence having a sequence identity of atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably ofat least 70%, more preferably of at least 80%, even more preferably atleast 85%, even more preferably of at least 90% and most preferably ofat least 95% or even 97%, with any one of the nucleic acid sequencesaccording to SEQ ID NOs: 34-37, 45-52, 60-63, 71-78, 86-89, 97-104,112-115, 123-130, 138-141, 149-156, 164-167, 175-182, 190-193, 201-208,216-219, 227-234, 606-609, 632-635, 658-661, 684-687, 710-713, 736-739,762-765, 788-791, 617-624, 643-650, 669-676, 695-702, 721-728, 747-754,773-780, 799-806, 840-843, 866-869, 892-895, 918-921, 944-947, 970¬-973,996-999, 1022-1025, 851-858, 877-884, 903-910, 929-936, 955-962,981-988, 1007-1014, 1033-1040, 1074-1077, 1100-1103, 1126-1129,1152-1155, 1178-1181, 1204-1207, 1230-1233, 1256-1259, 1085-1092,1111-1118, 1137-1144, 1163-1170, 1189-1196, 1215-1222, 1241-1248,1267-1274 (as defined in Table 1 and Table 1B) or a fragment or variantof any one of these sequences.

According to another preferred embodiment, the (pharmaceutical)composition or the vaccine according to the invention comprises at leastone nucleic acid comprising at least one nucleic acid sequence of thepresent invention, wherein the at least one coding sequence (encoding atleast one Hendra virus antigenic peptide or protein, and, optionally, asecretory signal sequence) of the at least one nucleic acid sequencecomprises or consists of a nucleic acid sequence having a sequenceidentity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,preferably of at least 70%, more preferably of at least 80%, even morepreferably at least 85%, even more preferably of at least 90% and mostpreferably of at least 95% or even 97%, with any one of the mRNAsequences according to SEQ ID NOs: 1282-1285, 1293-1300, 1308-1311,1319-1326, 1334-1337, 1345-1352, 1360-1363, 1371-1378, 1386-1389,1397-1404, 1412-1415, 1423-1430, 1438-1441, 1464-1467, 1490-1493,1449-1456, 1475-1482, 1501-1508 (as defined in Table 5) or a fragment orvariant of any one of these sequences.

In the context of the present invention, the (pharmaceutical)composition or vaccine may encode one or more of the Hendra virusantigenic proteins or peptides as defined herein or a fragment orvariant thereof.

The (pharmaceutical) composition or vaccine according to the inventionmay thus comprise at least one nucleic acid comprising at least onenucleic acid sequence comprising at least one coding region, encoding atleast one Hendra virus antigenic peptide or protein, particularly, atleast one Hendra virus protein selected from Hendra virus RNA-directedRNA polymerase (L), Hendra virus fusion protein (F), Hendra virusnon-structural protein (V), Hendra virus glycoprotein (G), Hendra virusnucleoprotein (N), Hendra virus matrix protein (M), Hendra virusphosphoprotein (P), Hendra virus protein C, and Hendra virus protein W,a fragment or variant thereof, wherein the at least one coding region ofthe at least one nucleic acid sequence encodes one specific Hendra virusantigenic peptide or protein as defined herein or a fragment or avariant thereof.

According to a further preferred embodiment, the (pharmaceutical)composition or the vaccine according to the invention comprises at leastone nucleic acid of the present invention, wherein the at least onecoding sequence of the at least one nucleic acid sequence encodes atleast one Nipah virus peptide or protein selected from Nipah virusRNA-directed RNA polymerase (L), Nipah virus fusion protein (F), Nipahvirus non-structural protein (V), Nipah virus glycoprotein (G), Nipahvirus nucleoprotein (N), Nipah virus matrix protein (M), Nipah virusphosphoprotein (P), Nipah virus protein C, and Nipah virus protein W, aswell as to fragments or variants of all these proteins.

In a particularly preferred embodiment, the (pharmaceutical) compositionor the vaccine according to the invention comprises at least one nucleicacid of the present invention, wherein the at least one coding sequenceof the at least one nucleic acid sequence encodes at least one Nipahvirus peptide or protein selected from Nipah virus fusion protein (F)and Nipah virus glycoprotein (G) (and, optionally, a secretory signalsequence) as well as to fragments or variants of all these proteins,preferably proteins or peptides according to SEQ ID NOs: 1-7, 12-18,573-579, 584-590, 807-813, 818-824, 1041-1047, 1052-1058, 1513-1515 or ahomolog, fragment or variant of any of these sequences (see Table 2 andTable 2B, column “A”).

Preferably, the (pharmaceutical) composition or the vaccine according tothe invention comprises at least one nucleic acid of the presentinvention, wherein the at least one coding sequence of the at least onenucleic acid sequence encodes at least one Nipah virus peptide orprotein selected from Nipah virus fusion protein (F) and Nipah virusglycoprotein (G) (and, optionally, a secretory signal sequence) as wellas to fragments or variants of all these proteins, wherein the Nipahvirus peptide or protein preferably comprises or consists of an aminoacid sequence having a sequence identity of at least 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99%, preferably of at least 70%, morepreferably of at least 80%, even more preferably at least 85%, even morepreferably of at least 90% and most preferably of at least 95% or even97%, with any one of the amino acid sequences according to SEQ ID NOs:1-7, 12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047, 1052-1058, ora homolog, fragment or variant of any of these sequences (see Table 2and Table 2B, column “A”) fragment or variant of any one of thesesequences.

More preferably, the (pharmaceutical) composition or the vaccineaccording to the invention comprises at least one nucleic acidcomprising at least one nucleic acid sequence of the present invention,wherein the at least one coding sequence of the at least one nucleicacid sequence comprises or consists of a nucleic acid sequence encodingat least one Nipah virus peptide or protein (and, optionally, asecretory signal sequence) preferably comprises or consists of an aminoacid sequence having a sequence identity of at least 80% with any one ofthe amino acid sequences according to SEQ ID NOs: 1-7, 12-18, 573-579,584-590, 807-813, 818-824, 1041-1047, 1052-1058, 1513-1515 or a homolog,fragment or variant of any of these sequences (see Table 2 and Table 2B,column “A”) fragment or variant of any one of these sequences.

In preferred embodiments, the (pharmaceutical) composition or thevaccine according to the invention comprises at least one nucleic acidcomprising at least one nucleic acid sequence of the present invention,wherein the at least one coding sequence (encoding at least one Nipahvirus antigenic peptide or protein, and, optionally, a secretory signalsequence) of the at least one nucleic acid sequence comprises orconsists of any one of the nucleic acid sequences according to SEQ IDNOs: 27-33, 38-44, 53-59, 64-70, 79-85, 90-96, 105-111, 116-122,131-137, 142-148, 157-163, 168-174, 183-189, 194-200, 209-215, 220-226,599-605, 625-631, 651-657, 677-683, 703-709, 729-735, 755-761, 781-787,610-616, 636-642, 662-668, 688-694, 714-720, 740-746, 766-772, 792-798,833-839, 859-865, 885-891, 911-917, 937-943, 963-969, 989-995,1015-1021, 844-850, 870-876, 896-902, 922-928, 948-954, 974-980,1000-1006, 1026-1032, 1067-1073, 1093-1099, 1119-1125, 1145-1151,1171-1177, 1197-1203, 1223-1229, 1249-1255, 1078-1084, 1104-1110,1130-1136, 1156-1162, 1182-1188, 1208-1214, 1234-1240, 1260-1266,1516-1539 (as defined in Table 2 and Table 2B) or a fragment or variantof any one of these sequences.

According to another embodiment, the (pharmaceutical) composition or thevaccine according to the invention comprises at least one nucleic acidcomprising at least one nucleic acid sequence of the present invention,wherein the at least one coding sequence (encoding at least one Nipahvirus antigenic peptide or protein, and, optionally, a secretory signalsequence) of the at least one nucleic acid sequence comprises orconsists of a nucleic acid sequence having a sequence identity of atleast 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, preferably ofat least 70%, more preferably of at least 80%, even more preferably atleast 85%, even more preferably of at least 90% and most preferably ofat least 95% or even 97%, with any one of the nucleic acid sequencesaccording to SEQ ID NOs: 27-33, 38-44, 53-59, 64-70, 79-85, 90-96,105-111, 116-122, 131-137, 142-148, 157-163, 168-174, 183-189, 194-200,209-215, 220-226, 599-605, 625-631, 651-657, 677-683, 703-709, 729-735,755-761, 781-787, 610-616, 636-642, 662-668, 688-694, 714-720, 740-746,766-772, 792-798, 833-839, 859-865, 885-891, 911-917, 937-943, 963-969,989-995, 1015-1021, 844-850, 870-876, 896-902, 922-928, 948-954,974-980, 1000-1006, 1026-1032, 1067-1073, 1093-1099, 1119-1125,1145-1151, 1171-1177, 1197-1203, 1223-1229, 1249-1255, 1078-1084,1104-1110, 1130-1136, 1156-1162, 1182-1188, 1208-1214, 1234-1240,1260-1266, 1516-1539 (as defined in Table 2 and Table 2B) or a fragmentor variant of any one of these sequences.

According to another preferred embodiment, the (pharmaceutical)composition or the vaccine according to the invention comprises at leastone nucleic acid comprising at least one nucleic acid sequence of thepresent invention, wherein the at least one coding sequence (encoding atleast one Nipah virus antigenic peptide or protein and, optionally, asecretory signal sequence) of the at least one nucleic acid sequencecomprises or consists of a nucleic acid sequence having a sequenceidentity of at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%,preferably of at least 70%, more preferably of at least 80%, even morepreferably at least 85%, even more preferably of at least 90% and mostpreferably of at least 95% or even 97%, with any one of the mRNAsequences according to SEQ ID NOs: 1275-1281, 1286-1292, 1301-1307,1312-1318, 1327-1333, 1338-1344, 1353-1359, 1364-1370, 1379-1385,1390-1396, 1405-1411, 1416-1422, 1431-1437, 1457-1463, 1483-1489,1442-1448, 1468-1474, 1494-1500, 1540-1548 (as defined in Table 6) or afragment or variant of any one of these sequences

In the context of the present invention, the (pharmaceutical)composition or vaccine may encode one or more of the Nipah virusantigenic proteins or peptides as defined herein or a fragment orvariant thereof.

The (pharmaceutical) composition or vaccine according to the inventionmay thus comprise at least one nucleic acid comprising at least onenucleic acid sequence comprising at least one coding region, encoding atleast one Nipah virus antigenic peptide or protein, particularly, atleast one Nipah virus protein selected from Nipah virus RNA-directed RNApolymerase (L), Nipah virus fusion protein (F), Nipah virusnon-structural protein (V), Nipah virus glycoprotein (G), Nipah virusnucleoprotein (N), Nipah virus matrix protein (M), Nipah virusphosphoprotein (P), Nipah virus protein C, and Nipah virus protein W, afragment or variant thereof, wherein the at least one coding region ofthe at least one nucleic acid sequence encodes one specific Nipah virusantigenic peptide or protein as defined herein or a fragment or avariant thereof.

Alternatively, the (pharmaceutical) composition or vaccine of thepresent invention may comprise at least one nucleic acid comprising atleast one nucleic acid sequence according to the invention, wherein theat least one nucleic acid sequence encodes at least two, three, four,five, six, seven, eight, nine, ten, eleven or twelve distinctHenipavirus and/or Hendra virus and/or Nipah virus antigenic peptides orproteins as defined herein or a fragment or variant thereof.

In this context it is particularly preferred that the at least onenucleic acid comprised in the (pharmaceutical) composition or vaccine isa bi- or multicistronic nucleic acid, particularly a bi- ormulticistronic nucleic acid as defined herein, which encodes the atleast two, three, four, five, six, seven, eight, nine, ten, eleven ortwelve distinct Henipavirus and/or Hendra virus and/or Nipah viruspeptides or proteins derived from a protein of a Henipavirus and/orHendra virus and/or Nipah virus. Mixtures between these embodiments arealso envisaged, such as compositions comprising more than one nucleicacid sequences, wherein at least one nucleic acid sequence may bemonocistronic, while at least one other nucleic acid sequence may be bi-or multicistronic.

The (pharmaceutical) composition or vaccine according to the presentinvention, preferably the at least one coding sequence of the nucleicacid sequence comprised therein, may thus comprise any combination ofthe nucleic acid sequences as defined herein.

Preferably, the (pharmaceutical) composition or vaccine comprises aplurality or more than one of the nucleic sequences according to theinvention, wherein each nucleic acid sequence comprises at least onecoding region encoding at least one antigenic peptide or protein derivedfrom a protein of a Henipavirus and/or Hendra virus and/or Nipah virusor a fragment or variant thereof.

In a particularly preferred embodiment, the composition comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different artificialnucleic acids each encoding at least one antigenic peptide or proteinderived from genetically the same Henipavirus and/or Hendra virus and/orNipah virus or a fragment or variant thereof.

In another preferred embodiment each nucleic acid sequence encodes atleast one different Henipavirus and/or Hendra virus and/or Nipah virusantigenic peptide or protein derived from proteins of differentHenipavirus and/or Hendra virus and/or Nipah virus or a fragment orvariant thereof.

In a particularly preferred embodiment, the composition comprises atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more different artificialnucleic acids each encoding at least one peptide or protein derived froma different Henipavirus and/or Hendra virus and/or Nipah virus or afragment or variant thereof.

In an embodiment, the composition comprises at least one artificialnucleic acid encoding at least one antigenic peptide or protein derivedfrom Hendra virus fusion protein (F) and/or at least one artificialnucleic acid encoding at least one antigenic peptide or protein derivedfrom Hendra virus glycoprotein (G) and/or at least one artificialnucleic acid encoding at least one antigenic peptide or protein derivedfrom Nipah virus fusion protein (F) and/or at least one artificialnucleic acid encoding at least one antigenic peptide or protein derivedfrom Nipah virus glycoprotein (G) or a fragment or variant thereof.

In a specific embodiment, the composition comprises at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Hendra virus fusion protein (F) and at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Hendra virus glycoprotein (G) or a fragment orvariant thereof.

In a specific embodiment, the composition comprises at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Nipah virus fusion protein (F) and at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Nipah virus glycoprotein (G) or a fragment orvariant thereof.

In a specific embodiment, the composition comprises at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Nipah virus fusion protein (F) and at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Hendra virus glycoprotein (G) or a fragment orvariant thereof.

In a specific embodiment, the composition comprises at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Nipah virus fusion protein (F) and at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Hendra virus fusion protein (F) or a fragment orvariant thereof.

In a specific embodiment, the composition comprises at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Nipah virus glycoprotein (G) and at least oneartificial nucleic acid encoding at least one antigenic peptide orprotein derived from Hendra virus glycoprotein (G) or a fragment orvariant thereof.

Complexation and Formulation:

In a preferred embodiment of the composition according to the invention,the at least one nucleic acid comprising at least one nucleic acidsequence according to the invention is complexed with one or morecationic or polycationic compounds, preferably with cationic orpolycationic polymers, cationic or polycationic peptides or proteins,e.g. protamine, cationic or polycationic polysaccharides and/or cationicor polycationic lipids.

According to a preferred embodiment, the at least one nucleic acid ofthe composition according to the present invention may be complexed withlipids to form one or more liposomes, lipoplexes, or lipidnanoparticles. Therefore, in one embodiment, the inventive compositioncomprises liposomes, lipoplexes, and/or lipid nanoparticles comprisingthe at least one nucleic acid, preferably RNA, more preferably mRNA.

Lipid-based formulations have been increasingly recognized as one of themost promising delivery systems for nucleic acids, particularly of RNA,due to their biocompatibility and their ease of large-scale production.Cationic lipids have been widely studied as synthetic materials fordelivery of RNA. After mixing together, nucleic acids are condensed bycationic lipids to form lipid/nucleic acid complexes known aslipoplexes. These lipid complexes are able to protect genetic materialfrom the action of nucleases and deliver it into cells by interactingwith the negatively charged cell membrane. Lipoplexes can be prepared bydirectly mixing positively charged lipids at physiological pH withnegatively charged nucleic acids.

Conventional liposomes consist of a lipid bilayer that can be composedof cationic, anionic, or neutral (phospho)lipids and cholesterol, whichencloses an aqueous core. Both the lipid bilayer and the aqueous spacecan incorporate hydrophobic or hydrophilic compounds, respectively.Liposome characteristics and behaviour in vivo can be modified byaddition of a hydrophilic polymer coating, e.g. polyethylene glycol(PEG), to the liposome surface to confer steric stabilization.Furthermore, liposomes can be used for specific targeting by attachingligands (e.g., antibodies, peptides, and carbohydrates) to its surfaceor to the terminal end of the attached PEG chains.

Liposomes are colloidal lipid-based and surfactant-based deliverysystems composed of a phospholipid bilayer surrounding an aqueouscompartment. They may present as spherical vesicles and can range insize from 20 nm to a few microns. Cationic lipid-based liposomes areable to complex with negatively charged nucleic acids via electrostaticinteractions, resulting in complexes that offer biocompatibility, lowtoxicity, and the possibility of the large-scale production required forin vivo clinical applications. Liposomes can fuse with the plasmamembrane for uptake; once inside the cell, the liposomes are processedvia the endocytic pathway and the genetic material is then released fromthe endosome/carrier into the cytoplasm. Liposomes have long beenperceived as drug delivery vehicles because of their superiorbiocompatibility, given that liposomes are basically analogs ofbiological membranes, and can be prepared from both natural andsynthetic phospholipids.

Cationic liposomes have been traditionally the most commonly usednon-viral delivery systems for oligonucleotides, including plasmid DNA,antisense oligos, and siRNA/small hairpin RNA-shRNA). Cationic lipids,such as DOTAP, (1,2-dioleoyl-3-trimethylammonium-propane) and DOTMA(N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethyl-ammonium methyl sulfate)can form complexes or lipoplexes with negatively charged nucleic acidsto form nanoparticles by electrostatic interaction, providing high invitro transfection efficiency. Furthermore, neutral lipid-basednanoliposomes for RNA delivery as e.g. neutral1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)-based nanoliposomeswere developed.

Therefore, in one embodiment the at least one nucleic acid, preferablythe RNA of the composition according to the present invention iscomplexed with cationic lipids and/or neutral lipids and thereby formsliposomes, lipid nanoparticles, lipoplexes or neutral lipid-basednanoliposomes.

In the context of the present invention, the term “lipid nanoparticle”,also referred to as “LNP”, is not restricted to any particularmorphology, and includes any morphology generated when a cationic lipidand optionally one or more further lipids are combined, e.g. in anaqueous environment and/or in the presence of an RNA. For example, aliposome, a lipid complex, a lipoplex, an emulsion, a micelle, a lipidicnanocapsule, a nanosuspension and the like are within the scope of alipid nanoparticle (LNP).

LNPs typically comprise a cationic lipid and one or more excipientselected from neutral lipids, charged lipids, steroids and polymerconjugated lipids (e.g. PEGylated lipid). The nucleic acid may beencapsulated in the lipid portion of the LNP or an aqueous spaceenveloped by some or the entire lipid portion of the LNP. The RNA or aportion thereof may also be associated and complexed with the LNP. AnLNP may comprise any lipid capable of forming a particle to which thenucleic acids are attached, or in which the one or more nucleic acidsare encapsulated. Preferably, the LNP comprising nucleic acids comprisesone or more cationic lipids, and one or more stabilizing lipids.Stabilizing lipids include neutral lipids and PEGylated lipids.

In one embodiment, the LNP consists essentially of (i) at least onecationic lipid; (ii) a neutral lipid; (iii) a sterol, e.g., cholesterol;and (iv) a PEG-lipid, e.g. PEG-DMG or PEG-cDMA, in a molar ratio ofabout 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol; 0.5-15%PEG-lipid.

In that context, a preferred sterol is cholesterol. The sterol can beabout 10 mol % to about 60 mol % or about 25 mol % to about 40 mol % ofthe lipid particle. In one embodiment, the sterol is about 10, 15, 20,25, 30, 35, 40, 45, 50, 55, or about 60 mol % of the total lipid presentin the lipid particle. In another embodiment, the LNPs include fromabout 5% to about 50% on a molar basis of the sterol, e.g., about 15% toabout 45%, about 20% to about 40%, about 48%, about 40%, about 38.5%,about 35%, about 34.4%, about 31.5% or about 31% on a molar basis (basedupon 100% total moles of lipid in the lipid nanoparticle).

The cationic lipid of an LNP may be cationisable, i.e. it becomesprotonated as the pH is lowered below the pK of the ionizable group ofthe lipid, but is progressively more neutral at higher pH values. At pHvalues below the pK, the lipid is then able to associate with negativelycharged nucleic acids. In certain embodiments, the cationic lipidcomprises a zwitterionic lipid that assumes a positive charge on pHdecrease.

The LNP may comprise any further cationic or cationisable lipid, i.e.any of a number of lipid species which carry a net positive charge at aselective pH, such as physiological pH.

Such lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (DODAC);N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA);N,N-distearyl-N,N-dimethylammonium bromide (DDAB);N-(2,3dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP);3-(N—(N′,N′dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol),N-(1-(2,3-dioleoyloxy)propyl)N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (DOSPA), dioctadecylamidoglycyl carboxyspermine (DOGS),1,2-dioleoyl-3-dimethylammonium propane (DODAP),N,N-dimethyl-2,3-dioleoyloxy)propylamine (DODMA), andN-(1,2dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (DMRIE). In some aspects, the lipid is selected from the groupconsisting of 98N12-5, C12-200, and ckk-E12.

In some embodiments, the lipid is selected from the group consisting of98N12-5, C12-200, and ckk-E12.

In one embodiment, the nucleic acids may be formulated in anaminoalcohol lipidoid. Aminoalcohol lipidoids which may be used in thepresent invention may be prepared by the methods described in U.S. Pat.No. 8,450,298, herein incorporated by reference in its entirety.Suitable ionizable lipids can also be the compounds as disclosed inTables 1, 2 and 3 and claims 1-24 of International Publication No. WO2017/075531 A1, hereby incorporated by reference in its entirety. Inanother embodiment, ionizable lipids can also be the compounds asdisclosed in International Publication No. WO 2015/074085 A1 (i.e.ATX-001 to ATX-032 or the compounds as mentioned in claims 1-26), U.S.Appl. Nos. 61/905,724 and Ser. No. 15/614,499 or U.S. Pat. Nos.9,593,077 and 9,567,296 hereby incorporated by reference in theirentirety.

Additionally, a number of commercial preparations of cationic lipids areavailable which can be used in the present invention. These include, forexample, LIPOFECTIN® (commercially available cationic liposomescomprising DOTMA and 1,2-dioleoyl-sn-3phosphoethanolamine (DOPE), fromGIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially availablecationic liposomes comprisingN-(1-(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM®(commercially available cationic lipids comprisingdioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from PromegaCorp., Madison, Wis.). The following lipids are cationic and have apositive charge at below physiological pH: DODAP, DODMA, DMDMA,1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA).

The further cationic lipid may also be an amino lipid. Representativeamino lipids include, but are not limited to,1,2-dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-dilinoleyoxy-3morpholinopropane (DLin-MA),1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-linoleoyl-2-linoleyloxy-3dimethylaminopropane (DLin-2-DMAP),1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ),3-(N,Ndilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-dioleylamino)-1,2-propanediol (DOAP),1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA),2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA);dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3(US20100324120).

Other suitable (cationic) lipids are disclosed in WO2009/086558,WO2009/127060, WO2010/048536, WO2010/054406, WO2010/088537,WO2010/129709, WO2011/153493, US2011/0256175, US2012/0128760,US2012/0027803, and U.S. Pat. No. 8,158,601. In that context, thedisclosures of WO2009/086558, WO2009/127060, WO2010/048536,WO2010/054406, WO2010/088537, WO2010/129709, WO2011/153493,US2011/0256175, US2012/0128760, US2012/0027803, and U.S. Pat. No.8,158,601 are incorporated herewith by reference.

The amount of the permanently cationic lipid or lipidoid may be selectedtaking the amount of the nucleic acid cargo into account. In oneembodiment, these amounts are selected such as to result in an N/P ratioof the nanoparticle(s) or of the composition in the range from about 0.1to about 20. In this context, the N/P ratio is defined as the mole ratioof the nitrogen atoms (“N”) of the basic nitrogen-containing groups ofthe lipid or lipidoid to the phosphate groups (“P”) of the RNA which isused as cargo. The N/P ratio may be calculated on the basis that, forexample, 1 μg RNA typically contains about 3 nmol phosphate residues,provided that the RNA exhibits a statistical distribution of bases. The“N”-value of the lipid or lipidoid may be calculated on the basis of itsmolecular weight and the relative content of permanently cationic and—ifpresent—cationisable groups.

In certain embodiments, the LNP comprises one or more additional lipidswhich stabilize the formation of particles during their formation.

Suitable stabilizing lipids include neutral lipids and anionic lipids.The term “neutral lipid” refers to any one of a number of lipid speciesthat exist in either an uncharged or neutral zwitterionic form atphysiological pH. Representative neutral lipids includediacylphosphatidylcholines, diacylphosphatidylethanolamines, ceramides,sphingomyelins, dihydro sphingomyelins, cephalins, and cerebrosides.

Exemplary neutral lipids include, for example,distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine(DOPC), dipalmitoylphosphatidylcholine (DPPC),dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol(DPPG), dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE) anddioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE, 1-stearioyl-2-oleoylphosphatidyethanolamine (SOPE), and 1,2-dielaidoyl-sn-glycero-3-phophoethanolamine(transDOPE). In one embodiment, the neutral lipid is1,2-distearoyl-sn-glycero-3phosphocholine (DSPC).

In some embodiments, the LNPs comprise a neutral lipid selected fromDSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In various embodiments, themolar ratio of the cationic lipid to the neutral lipid ranges from about2:1 to about 8:1.

LNP in vivo characteristics and behavior can be modified by addition ofa hydrophilic polymer coating, e.g. polyethylene glycol (PEG), to theLNP surface to confer steric stabilization. Furthermore, LNPs can beused for specific targeting by attaching ligands (e.g. antibodies,peptides, and carbohydrates) to its surface or to the terminal end ofthe attached PEG chains (e.g. via PEGylated lipids).

In some embodiments, the LNPs comprise a polymer conjugated lipid. Theterm “polymer conjugated lipid” refers to a molecule comprising both alipid portion and a polymer portion. An example of a polymer conjugatedlipid is a PEGylated lipid. The term “PEGylated lipid” refers to amolecule comprising both a lipid portion and a polyethylene glycolportion. PEGylated lipids are known in the art and include1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-s-DMG)and the like.

In certain embodiments, the LNP comprises an additional,stabilizing-lipid which is a polyethylene glycol-lipid (PEGylatedlipid). Suitable polyethylene glycol-lipids include PEG-modifiedphosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modifiedceramides (e.g. PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines,PEG-modified diacylglycerols, PEG-modified dialkylglycerols.Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA,and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid isN-[(methoxy poly(ethyleneglycol)2000)carbamyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). Inone embodiment, the polyethylene glycol-lipid is PEG-c-DOMG). In otherembodiments, the LNPs comprise a PEGylated diacylglycerol (PEG-DAG) suchas 1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol (PEG-DMG),a PEGylated phosphatidylethanoloamine (PEG-PE), a PEG succinatediacylglycerol (PEG-S-DAG) such as4-O-(2′,3′-di(tetradecanoyloxy)propyl-1-O-(ω-methoxy(polyethoxy)ethyl)butanedioate(PEG-S-DMG), a PEGylated ceramide (PEG-cer), or a PEGdialkoxypropylcarbamate such asω-methoxy(polyethoxy)ethyl-N-(2,3-di(tetradecanoxy)propyl)carbamate or2,3-di(tetradecanoxy)propyl-N-(ω-methoxy(polyethoxy)ethyl)carbamate.

Further examples of PEG-lipids suitable in that context are provided inUS20150376115A1 and WO2015199952, each of which is incorporated byreference in its entirety.

In some embodiments, LNPs include less than about 3, 2, or 1 molepercent of PEG or PEG-modified lipid, based on the total moles of lipidin the LNP. In further embodiments, LNPs comprise from about 0.1% toabout 20% of the PEG-modified lipid on a molar basis, e.g., about 0.5 toabout 10%, about 0.5 to about 5%, about 10%, about 5%, about 3.5%, about3%, about 2.5%, about 2%, about 1.5%, about 1%, about 0.5%, or about0.3% on a molar basis (based on 100% total moles of lipids in the LNP).

In various embodiments, the molar ratio of the cationic lipid to thePEGylated lipid ranges from about 100:1 to about 25:1.

The total amount of nucleic acid, particularly the RNA in the lipidnanoparticles varies and may be defined depending on the e.g. RNA tototal lipid w/w ratio. In one embodiment of the invention the RNA tototal lipid ratio is less than 0.06 w/w, preferably between 0.03 w/w and0.04 w/w.

In a preferred embodiment, the composition according to the inventioncomprises the nucleic acid comprising at least one nucleic acid sequenceaccording to the invention that is formulated together with a cationicor polycationic compound and/or with a polymeric carrier. Accordingly,in a further embodiment of the invention, it is preferred that thenucleic acid as defined herein or any other nucleic acid comprised inthe inventive (pharmaceutical) composition or vaccine is associated withor complexed with a cationic or polycationic compound or a polymericcarrier, optionally in a weight ratio selected from a range of about 6:1(w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) toabout 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1(w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably aratio of about 3:1 (w/w) to about 2:1 (w/w) of mRNA or nucleic acid tocationic or polycationic compound and/or with a polymeric carrier; oroptionally in a nitrogen/phosphate (N/P) ratio of mRNA or nucleic acidto cationic or polycationic compound and/or polymeric carrier in therange of about 0.1-10, preferably in a range of about 0.3-4 or 0.3-1,and most preferably in a range of about 0.5-1 or 0.7-1, and even mostpreferably in a range of about 0.3-0.9 or 0.5-0.9. More preferably, theN/P ratio of the at least one mRNA to the one or more polycations is inthe range of about 0.1 to 10, including a range of about 0.3 to 4, ofabout 0.5 to 2, of about 0.7 to 2 and of about 0.7 to 1.5.

Therein, the nucleic acid as defined herein or any other nucleic acidcomprised in the (pharmaceutical) composition or vaccine according tothe invention can also be associated with a vehicle, transfection orcomplexation agent for increasing the transfection efficiency and/or theimmunostimulatory properties of the nucleic acid according to theinvention or of optionally comprised further included nucleic acids.

Cationic or polycationic compounds, being particularly preferred agentsin this context include protamine, nucleoline, spermine or spermidine,or other cationic peptides or proteins, such as poly-L-lysine (PLL),poly-arginine, basic polypeptides, cell penetrating peptides (CPPs),including HIV-binding peptides, HIV-1 Tat (HIV), Tat-derived peptides,Penetratin, VP22 derived or analog peptides, HSV VP22 (Herpes simplex),MAP, KALA or protein transduction domains (PTDs), PpT620, prolin-richpeptides, arginine-rich peptides, lysine-rich peptides, MPG-peptide(s),Pep-1, L-oligomers, Calcitonin peptide(s), Antennapedia-derived peptides(particularly from Drosophila antennapedia), pAntp, plsl, FGF,Lactoferrin, Transportan, Buforin-2, Bac715-24, SynB, SynB(1), pVEC,hCT-derived peptides, SAP, or histones. More preferably, the mRNAaccording to the invention is complexed with one or more polycations,preferably with protamine or oligofectamine, most preferably withprotamine. In this context protamine is particularly preferred.

Additionally, preferred cationic or polycationic proteins or peptidesmay be selected from the following proteins or peptides having thefollowing total formula (III):(Arg)l;(Lys)m;(His)n;(Orn)o;(Xaa)x,  (formula (III))wherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, provided that the overall content of Arg, Lys, His and Ornrepresents at least 50% of all amino acids of the oligopeptide; and Xaamay be any amino acid selected from native (=naturally occurring) ornon-native amino acids except of Arg, Lys, His or Orn; and x may be anynumber selected from 0, 1, 2, 3 or 4, provided, that the overall contentof Xaa does not exceed 50% of all amino acids of the oligopeptide.Particularly preferred cationic peptides in this context are e.g. Arg7,Arg8, Arg9, H3R9, R9H3, H3R9H3, YSSR9SSY, (RKH)4, Y(RKH)2R, etc. In thiscontext the disclosure of WO 2009/030481 is incorporated herewith byreference.

Preferred cationic or polycationic proteins or peptides may be derivedfrom formula Cys{(Arg)_(l); (Lys)m; (His)_(n); (Orn)_(o); (Xaa)_(x)}Cysor {(Arg)_(l); (Lys)_(m); (His)_(n); (Orn)_(o); (Xaa)_(x)} of the patentapplication WO2009/030481 or WO2011/026641, the disclosure ofWO2009/030481 and WO2011/026641 relating thereto are incorporatedherewith by reference. In a preferred embodiment, the cationic orpolycationic proteins or peptides comprises CHHHHHHRRRRHHHHHHC (SEQ IDNO: 309), CR₁₂C (SEQ ID NO: 306), CR₁₂ (SEQ ID NO: 307) or WR₁₂C (SEQ IDNO: 308).

Further preferred cationic or polycationic compounds, which can be usedas transfection or complexation agent may include cationicpolysaccharides, for example chitosan, polybrene, cationic polymers,e.g. polyethyleneimine (PEI), cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected from a cationic polymer as mentioned above) and of one ormore hydrophilic or hydrophobic blocks (e.g. polyethyleneglycole); etc.

According to a preferred embodiment, the composition of the presentinvention comprises the nucleic acid as defined herein, preferably anRNA, and a polymeric carrier. A polymeric carrier used according to theinvention might be a polymeric carrier formed by disulfide-crosslinkedcationic components. The disulfide-crosslinked cationic components maybe the same or different from each other. The polymeric carrier can alsocontain further components. It is also particularly preferred that thepolymeric carrier used according to the present invention comprisesmixtures of cationic peptides, proteins or polymers and optionallyfurther components as defined herein, which are crosslinked by disulfidebonds as described herein. In this context, the disclosure of WO2012/013326 is incorporated herewith by reference.

In this context, the cationic components, which form basis for thepolymeric carrier by disulfide-crosslinkage, are typically selected fromany suitable cationic or polycationic peptide, protein or polymersuitable for this purpose, particular any cationic or polycationicpeptide, protein or polymer capable of complexing the mRNA as definedherein or a further nucleic acid comprised in the composition, andthereby preferably condensing the mRNA or the nucleic acid. The cationicor polycationic peptide, protein or polymer, is preferably a linearmolecule, however, branched cationic or polycationic peptides, proteinsor polymers may also be used.

Every disulfide-crosslinking cationic or polycationic protein, peptideor polymer of the polymeric carrier, which may be used to complex themRNA according to the invention or any further nucleic acid comprised inthe (pharmaceutical) composition or vaccine of the present inventioncontains at least one —SH moiety, most preferably at least one cysteineresidue or any further chemical group exhibiting an —SH moiety, capableof forming a disulfide linkage upon condensation with at least onefurther cationic or polycationic protein, peptide or polymer as cationiccomponent of the polymeric carrier as mentioned herein.

As defined above, the polymeric carrier, which may be used to complexthe nucleic acid of the present invention or any further nucleic acidcomprised in the (pharmaceutical) composition or vaccine according tothe invention may be formed by disulfide-crosslinked cationic (orpolycationic) components. Preferably, such cationic or polycationicpeptides or proteins or polymers of the polymeric carrier, whichcomprise or are additionally modified to comprise at least one —SHmoiety, are selected from, proteins, peptides and polymers as definedherein for complexation agent.

In a further particular embodiment, the polymeric carrier which may beused to complex the nucleic acid as defined herein or any furthernucleic acid comprised in the (pharmaceutical) composition or vaccineaccording to the invention may be selected from a polymeric carriermolecule according to generic formula (IV):L-P¹—S—[S—P²—S]_(n)—S—P³-L  formula (IV)wherein,

-   P¹ and P³ are different or identical to each other and represent a    linear or branched hydrophilic polymer chain, each P¹ and P³    exhibiting at least one —SH-moiety, capable to form a disulfide    linkage upon condensation with component P², or alternatively with    (AA), (AA)_(x), or [(AA)_(x)]_(z) if such components are used as a    linker between P¹ and P² or P³ and P²) and/or with further    components (e.g. (AA), (AA)_(x), [(AA)_(x)]_(z) or L), the linear or    branched hydrophilic polymer chain selected independent from each    other from polyethylene glycol (PEG),    poly-N-(2-hydroxypropyl)methacrylamide,    poly-2-(methacryloyloxy)ethyl phosphorylcholines, poly(hydroxyalkyl    L-asparagine), poly(2-(methacryloyloxy)ethyl phosphorylcholine),    hydroxyethylstarch or poly(hydroxyalkyl L-glutamine), wherein the    hydrophilic polymer chain exhibits a molecular weight of about 1 kDa    to about 100 kDa, preferably of about 2 kDa to about 25 kDa; or more    preferably of about 2 kDa to about 10 kDa, e.g. about 5 kDa to about    25 kDa or 5 kDa to about 10 kDa;-   P² is a cationic or polycationic peptide or protein, e.g. as defined    above for the polymeric carrier formed by disulfide-crosslinked    cationic components, and preferably having a length of about 3 to    about 100 amino acids, more preferably having a length of about 3 to    about 50 amino acids, even more preferably having a length of about    3 to about 25 amino acids, e.g. a length of about 3 to 10, 5 to 15,    10 to 20 or 15 to 25 amino acids, more preferably a length of about    5 to about 20 and even more preferably a length of about 10 to about    20; or    -   is a cationic or polycationic polymer, e.g. as defined above for        the polymeric carrier formed by disulfide-crosslinked cationic        components, typically having a molecular weight of about 0.5 kDa        to about 30 kDa, including a molecular weight of about 1 kDa to        about 20 kDa, even more preferably of about 1.5 kDa to about 10        kDa, or having a molecular weight of about 0.5 kDa to about 100        kDa, including a molecular weight of about 10 kDa to about 50        kDa, even more preferably of about 10 kDa to about 30 kDa;    -   each P² exhibiting at least two —SH-moieties, capable to form a        disulfide linkage upon condensation with further components P²        or component(s) P¹ and/or P³ or alternatively with further        components (e.g. (AA), (AA)_(x), or [(AA)_(x)]_(z));-   —S—S— is a (reversible) disulfide bond (the brackets are omitted for    better readability), wherein S preferably represents sulphur or a    —SH carrying moiety, which has formed a (reversible) disulfide bond.    The (reversible) disulfide bond is preferably formed by condensation    of —SH-moieties of either components P¹ and P², P² and P², or P² and    P³, or optionally of further components as defined herein (e.g. L,    (AA), (AA)_(x), [(AA)_(x)]_(z), etc); The —SH-moiety may be part of    the structure of these components or added by a modification as    defined below;-   L is an optional ligand, which may be present or not, and may be    selected independent from the other from RGD, Transferrin, Folate, a    signal peptide or signal sequence, a localization signal or    sequence, a nuclear localization signal or sequence (NLS), an    antibody, a cell penetrating peptide, (e.g. TAT or KALA), a ligand    of a receptor (e.g. cytokines, hormones, growth factors etc), small    molecules (e.g. carbohydrates like mannose or galactose or synthetic    ligands), small molecule agonists, inhibitors or antagonists of    receptors (e.g. RGD peptidomimetic analogues), or any further    protein as defined herein, etc.;-   n is an integer, typically selected from a range of about 1 to 50,    preferably from a range of about 1, 2 or 3 to 30, more preferably    from a range of about 1, 2, 3, 4, or 5 to 25, or a range of about 1,    2, 3, 4, or 5 to 20, or a range of about 1, 2, 3, 4, or 5 to 15, or    a range of about 1, 2, 3, 4, or 5 to 10, including e.g. a range of    about 4 to 9, 4 to 10, 3 to 20, 4 to 20, 5 to 20, or 10 to 20, or a    range of about 3 to 15, 4 to 15, 5 to 15, or 10 to 15, or a range of    about 6 to 11 or 7 to 10. Most preferably, n is in a range of about    1, 2, 3, 4, or 5 to 10, more preferably in a range of about 1, 2, 3,    or 4 to 9, in a range of about 1, 2, 3, or 4 to 8, or in a range of    about 1, 2, or 3 to 7.

In this context, the disclosure of WO 2011/026641 is incorporatedherewith by reference. Each of hydrophilic polymers P1 and P3 typicallyexhibits at least one —SH-moiety, wherein the at least one —SH-moiety iscapable to form a disulfide linkage upon reaction with component P2 orwith component (AA) or (AA)x, if used as linker between P1 and P2 or P3and P2 as defined below and optionally with a further component, e.g. Land/or (AA) or (AA)x, e.g. if two or more —SH-moieties are contained.The following subformulae “P1-S—S—P2” and “P2-S—S—P3” within genericformula (IV) above (the brackets are omitted for better readability),wherein any of S, P1 and P3 are as defined herein, typically represent asituation, wherein one —SH-moiety of hydrophilic polymers P1 and P3 wascondensed with one —SH-moiety of component P2 of generic formula (IV)above, wherein both sulphurs of these —SH-moieties form a disulfide bond—S—S— as defined herein in formula (IV). These —SH-moieties aretypically provided by each of the hydrophilic polymers P1 and P3, e.g.via an internal cysteine or any further (modified) amino acid orcompound which carries a —SH moiety. Accordingly, the subformulae“P1-S—S—P2” and “P2-S—S—P3” may also be written as “P1-Cys-Cys-P2” and“P2-Cys-Cys-P3”, if the —SH— moiety is provided by a cysteine, whereinthe term “Cys-Cys” represents two cysteines coupled via a disulfidebond, not via a peptide bond. In this case, the term “—S—S—” in theseformulae may also be written as “—S-Cys”, as “-Cys-S” or as “-Cys-Cys-”.In this context, the term “-Cys-Cys-” does not represent a peptide bondbut a linkage of two cysteines via their —SH-moieties to form adisulfide bond. Accordingly, the term “-Cys-Cys-” also may be understoodgenerally as “-(Cys-S)—(S-Cys)-”, wherein in this specific case Sindicates the sulphur of the —SH-moiety of cysteine. Likewise, the terms“—S-Cys” and “—Cys-S” indicate a disulfide bond between a —SH containingmoiety and a cysteine, which may also be written as “—S—(S-Cys)” and“-(Cys-S)—S”. Alternatively, the hydrophilic polymers P1 and P3 may bemodified with a —SH moiety, preferably via a chemical reaction with acompound carrying a —SH moiety, such that each of the hydrophilicpolymers P1 and P3 carries at least one such —SH moiety. Such a compoundcarrying a —SH moiety may be e.g. an (additional) cysteine or anyfurther (modified) amino acid, which carries a —SH moiety. Such acompound may also be any non-amino compound or moiety, which contains orallows to introduce a —SH moiety into hydrophilic polymers P1 and P3 asdefined herein. Such non-amino compounds may be attached to thehydrophilic polymers P1 and P3 of formula (IV) of the polymeric carrieraccording to the present invention via chemical reactions or binding ofcompounds, e.g. by binding of a 3-thio propionic acid or thioimolane, byamide formation (e.g. carboxylic acids, sulphonic acids, amines, etc),by Michael addition (e.g maleinimide moieties, α,β-unsatured carbonyls,etc), by click chemistry (e.g. azides or alkines), by alkene/alkinemethatesis (e.g. alkenes or alkines), imine or hydrozone formation(aldehydes or ketons, hydrazins, hydroxylamins, amines), complexationreactions (avidin, biotin, protein G) or components which allow Sn-typesubstitution reactions (e.g halogenalkans, thiols, alcohols, amines,hydrazines, hydrazides, sulphonic acid esters, oxyphosphonium salts) orother chemical moieties which can be utilized in the attachment offurther components. A particularly preferred PEG derivate in thiscontext is alpha-Methoxy-omega-mercapto poly(ethylene glycol). In eachcase, the SH-moiety, e.g. of a cysteine or of any further (modified)amino acid or compound, may be present at the terminal ends orinternally at any position of hydrophilic polymers P1 and P3. As definedherein, each of hydrophilic polymers P1 and P3 typically exhibits atleast one —SH-moiety preferably at one terminal end, but may alsocontain two or even more —SH-moieties, which may be used to additionallyattach further components as defined herein, preferably furtherfunctional peptides or proteins e.g. a ligand, an amino acid component(AA) or (AA)x, antibodies, cell penetrating peptides or enhancerpeptides (e.g. TAT, KALA), etc.

In a particularly preferred embodiment, the polymeric carrier is apeptide polymer, preferably a polyethylene glycol/peptide polymercomprising HO-PEG₅₀₀₀-S—(S—CHHHHHHRRRRHHHHHHC—S—)₇—S-PEG₅₀₀₀-OH (peptidemonomer: SEQ ID NO: 309) and a lipid component, preferably a lipidoidcomponent, more preferably lipidoid 3-C12-OH.

The lipidoid 3-C12-OH

(as shown above) may be obtained by acylation of tris(2-aminoethyl)aminewith an activated lauric (C12) acid derivative, followed by reduction ofthe amide. Alternatively, it may be prepared by reductive amination withthe corresponding aldehyde. Lipidoid 3-C12-OH is prepared by addition ofthe terminal C12 alkyl epoxide with the same oligoamine according toLove et al., pp. 1864-1869, PNAS, vol. 107 (2010), no. 5 (cf. compoundC12 and compound 110 in FIG. 1 of Love et al.). In preferredembodiments, the peptide polymer comprising lipidoid 3-C12-OH asspecified above is used to complex the artificial nucleic acid of theinvention, in particular RNA, to form complexes having an N/P ratio fromabout 0.1 to about 20, or from about 0.2 to about 15, or from about 2 toabout 15, or from about 2 to about 12, wherein the N/P ratio is definedas the mole ratio of the nitrogen atoms of the basic groups of thecationic peptide or polymer to the phosphate groups of the artificialnucleic acid.

In another embodiment, the polymeric carrier comprises a lipidoidcompound according to formula Ia

wherein

-   -   R_(A) is independently selected for each occurrence an        unsubstituted, cyclic or acyclic, branched or unbranched C₁₋₂₀        aliphatic group; a substituted or unsubstituted, cyclic or        acyclic, branched or unbranched C₁₋₂₀ heteroaliphatic group; a        substituted or unsubstituted aryl; a substituted or        unsubstituted heteroaryl;

wherein at least one R_(A) is

-   -   R₅ is independently selected for each occurrence of from an        unsubstituted, cyclic or acyclic, branched or unbranched C₈₋₁₆        aliphatic; a substituted or unsubstituted aryl; or a substituted        or unsubstituted heteroaryl;    -   each occurrence of x is an integer from 1 to 10;    -   each occurrence of y is an integer from 1 to 10;        or a pharmaceutically acceptable salt thereof.

In that context, the disclosure of the PCT patent applicationPCT/EP2017/064059 is herewith incorporated by reference.

In other embodiments, the composition, which is preferably a(pharmaceutical) composition comprises at least one artificial nucleicacid as described herein, wherein the at least one artificial nucleicacid is complexed or associated with polymeric carriers and, optionally,with at least one lipid component as described in the PCT applicationsPCT/EP2017/064065, PCT/EP2017/064058. In this context, the disclosuresof PCT/EP2017/064065, and PCT/EP2017/064058 is herewith incorporated byreference.

Preferably, the inventive composition comprises at least one nucleicacid as defined herein, which is complexed with one or more polycations,and at least one free nucleic acid, wherein the at least one complexednucleic acid is preferably identical to the at least one free nucleicacid. In this context, it is particularly preferred that the compositionof the present invention comprises the nucleic acid, preferably the RNA,according to the invention that is complexed at least partially with acationic or polycationic compound and/or a polymeric carrier, preferablycationic proteins or peptides. In this context, the disclosure of WO2010/037539 and WO 2012/113513 is incorporated herewith by reference.Partially means that only a part of the nucleic acid as defined hereinis complexed in the composition according to the invention with acationic compound and that the rest of the nucleic acid as definedherein is (comprised in the inventive (pharmaceutical) composition orvaccine) in uncomplexed form (“free”). Preferably, the molar ratio ofthe complexed nucleic acid to the free nucleic acid is selected from amolar ratio of about 0.001:1 to about 1:0.001, including a ratio ofabout 1:1. More preferably the ratio of complexed nucleic acid to freenucleic acid (in the (pharmaceutical) composition or vaccine of thepresent invention) is selected from a range of about 5:1 (w/w) to about1:10 (w/w), more preferably from a range of about 4:1 (w/w) to about 1:8(w/w), even more preferably from a range of about 3:1 (w/w) to about 1:5(w/w) or 1:3 (w/w), and most preferably the ratio of complexed nucleicacid to free nucleic acid in the inventive pharmaceutical composition orvaccine is selected from a ratio of about 1:1 (w/w).

The complexed nucleic acid in the (pharmaceutical) composition orvaccine according to the present invention, is preferably preparedaccording to a first step by complexing the nucleic acid according tothe invention with a cationic or polycationic compound and/or with apolymeric carrier, preferably as defined herein, in a specific ratio toform a stable complex. In this context, it is highly preferable, that nofree cationic or polycationic compound or polymeric carrier or only anegligibly small amount thereof remains in the component of thecomplexed nucleic acid after complexing the nucleic acid. Accordingly,the ratio of the nucleic acid and the cationic or polycationic compoundand/or the polymeric carrier in the component of the complexed RNA istypically selected in a range so that the nucleic acid is entirelycomplexed and no free cationic or polycationic compound or polymericcarrier or only a negligibly small amount thereof remains in thecomposition.

Preferably the ratio of the nucleic acid, preferably the RNA as definedherein to the cationic or polycationic compound and/or the polymericcarrier, preferably as defined herein, is selected from a range of about6:1 (w/w) to about 0.25:1 (w/w), more preferably from about 5:1 (w/w) toabout 0.5:1 (w/w), even more preferably of about 4:1 (w/w) to about 1:1(w/w) or of about 3:1 (w/w) to about 1:1 (w/w), and most preferably aratio of about 3:1 (w/w) to about 2:1 (w/w). Alternatively, the ratio ofthe nucleic acid as defined herein to the cationic or polycationiccompound and/or the polymeric carrier, preferably as defined herein, inthe component of the complexed nucleic acid, may also be calculated onthe basis of the nitrogen/phosphate ratio (N/P-ratio) of the entirecomplex. In the context of the present invention, an N/P-ratio ispreferably in the range of about 0.1-10, preferably in a range of about0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regardingthe ratio of mRNA:cationic or polycationic compound and/or polymericcarrier, preferably as defined herein, in the complex, and mostpreferably in a range of about 0.7-1.5, 0.5-1 or 0.7-1, and even mostpreferably in a range of about 0.3-0.9 or 0.5-0.9, preferably providedthat the cationic or polycationic compound in the complex is a cationicor polycationic cationic or polycationic protein or peptide and/or thepolymeric carrier as defined above. In this specific embodiment thecomplexed mRNA as defined herein is also encompassed in the term“adjuvant component”.

In other embodiments, the composition according to the inventioncomprising the nucleic acid, preferably the mRNA as defined herein maybe administered naked without being associated with any further vehicle,transfection or complexation agent.

It has to be understood and recognized, that according to the presentinvention, the inventive composition may comprise at least one nakednucleic acid, particularly naked mRNA as defined herein and/or at leastone formulated/complexed mRNA as defined herein, wherein everyformulation and/or complexation as disclosed above may be used.

Adjuvants:

According to another embodiment, the (pharmaceutical) composition orvaccine according to the invention may comprise an adjuvant, which ispreferably added in order to enhance the immunostimulatory properties ofthe composition. In this context, an adjuvant may be understood as anycompound, which is suitable to support administration and delivery ofthe composition according to the invention. Furthermore, such anadjuvant may, without being bound thereto, initiate or increase animmune response of the innate immune system, i.e. a non-specific immuneresponse. In other words, when administered, the composition accordingto the invention typically initiates an adaptive immune response due toan NIPAH virus antigen as defined herein or a fragment or variantthereof, which is encoded by the at least one coding sequence of theinventive mRNA contained in the composition of the present invention.Additionally, the composition according to the invention may generate an(supportive) innate immune response due to addition of an adjuvant asdefined herein to the composition according to the invention.

Such an adjuvant may be selected from any adjuvant known to a skilledperson and suitable for the present case, i.e. supporting the inductionof an immune response in a mammal. Preferably, the adjuvant may beselected from the group consisting of, without being limited thereto,TDM, MDP, muramyl dipeptide, pluronics, alum solution, aluminiumhydroxide, ADJUMER™ (polyphosphazene); aluminium phosphate gel; glucansfrom algae; algammulin; aluminium hydroxide gel (alum); highlyprotein-adsorbing aluminium hydroxide gel; low viscosity aluminiumhydroxide gel; AF or SPT (emulsion of squalane (5%), Tween 80 (0.2%),Pluronic L121 (1.25%), phosphate-buffered saline, pH 7.4); AVRIDINE™(propanediamine); BAY R1005™((N-(2-deoxy-2-L-leucylamino-b-D-glucopyranosyl)-N-octadecyl-dodecanoyl-amidehydroacetate); CALCITRIOL™ (1-alpha,25-dihydroxy-vitamin D3); calciumphosphate gel; CAP™ (calcium phosphate nanoparticles); choleraholotoxin, cholera-toxin-A1-protein-A-D-fragment fusion protein,sub-unit B of the cholera toxin; CRL 1005 (block copolymer P1205);cytokine-containing liposomes; DDA (dimethyldioctadecylammoniumbromide); DHEA (dehydroepiandrosterone); DMPC(dimyristoylphosphatidylcholine); DMPG(dimyristoylphosphatidylglycerol); DOC/alum complex (deoxycholic acidsodium salt); Freund's complete adjuvant; Freund's incomplete adjuvant;gamma inulin; Gerbu adjuvant (mixture of: i)N-acetylglucosaminyl-(P1-4)-N-acetylmuramyl-L-alanyl-D-glutamine (GMDP),ii) dimethyldioctadecylammonium chloride (DDA), iii) zinc-L-proline saltcomplex (ZnPro-8); GM-CSF); GMDP(N-acetylglucosaminyl-(b1-4)-N-acetylmuramyl-L-alanyl-D-isoglutamine);imiquimod (1-(2-methypropyl)-1H-imidazo[4,5-c]quinoline-4-amine);ImmTherm™(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-glyceroldipalmitate); DRVs (immunoliposomes prepared fromdehydration-rehydration vesicles); interferon-gamma; interleukin-1beta;interleukin-2; interleukin-7; interleukin-12; ISCOMS™; ISCOPREP 7.0.3.™;liposomes; LOXORIBINE™ (7-allyl-8-oxoguanosine); LT oral adjuvant (E.coli labile enterotoxin-protoxin); microspheres and microparticles ofany composition; MF59™; (squalene-water emulsion); MONTANIDE ISA 51™(purified incomplete Freund's adjuvant); MONTANIDE ISA 720™(metabolisable oil adjuvant); MPL™ (3-Q-desacyl-4′-monophosphoryl lipidA); MTP-PE and MTP-PE liposomes((N-acetyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1,2-dipalmitoyl-sn-glycero-3-(hydroxyphosphoryloxy))-ethylamide,monosodium salt); MURAMETIDE™ (Nac-Mur-L-Ala-D-Gln-OCH3); MURAPALMITINE™and D-MURAPALMITINE™ (Nac-Mur-L-Thr-D-isoGln-sn-glyceroldipalmitoyl);NAGO (neuraminidase-galactose oxidase); nanospheres or nanoparticles ofany composition; NISVs (non-ionic surfactant vesicles); PLEURAN™(β-glucan); PLGA, PGA and PLA (homo- and co-polymers of lactic acid andglycolic acid; microspheres/nanospheres); PLURONIC L121™; PMMA(polymethyl methacrylate); PODDS™ (proteinoid microspheres);polyethylene carbamate derivatives; poly-rA: poly-rU (polyadenylicacid-polyuridylic acid complex); polysorbate 80 (Tween 80); proteincochleates (Avanti Polar Lipids, Inc., Alabaster, Ala.); STIMULON™(QS-21); Quil-A (Quil-A saponin); S-28463(4-amino-otec-dimethyl-2-ethoxymethyl-1H-imidazo[4,5c]quinoline-1-ethanol); SAF-1™ (“Syntex adjuvant formulation”); Sendaiproteoliposomes and Sendai-containing lipid matrices; Span-85 (sorbitantrioleate); Specol (emulsion of Marcol 52, Span 85 and Tween 85);squalene or Robane® (2,6,10,15,19,23-hexamethyltetracosan and2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexane);stearyltyrosine (octadecyltyrosine hydrochloride); Theramid®(N-acetylglucosaminyl-N-acetylmuramyl-L-Ala-D-isoGlu-L-Ala-dipalmitoxypropylamide);Theronyl-MDP (Termurtide™ or [thr 1]-MDP;N-acetylmuramyl-L-threonyl-D-isoglutamine); Ty particles (Ty-VLPs orvirus-like particles); Walter-Reed liposomes (liposomes containing lipidA adsorbed on aluminium hydroxide), and lipopeptides, including Pam3Cys,in particular aluminium salts, such as Adju-phos, Alhydrogel,Rehydragel; emulsions, including CFA, SAF, IFA, MF59, Provax, TiterMax,Montanide, Vaxfectin; copolymers, including Optivax (CRL1005), L121,Poloaxmer4010), etc.; liposomes, including Stealth, cochleates,including BIORAL; plant derived adjuvants, including QS21, Quil A,Iscomatrix, ISCOM; adjuvants suitable for costimulation includingTomatine, biopolymers, including PLG, PMM, Inulin; microbe derivedadjuvants, including Romurtide, DETOX, MPL, CWS, Mannose, CpG nucleicacid sequences, CpG7909, ligands of human TLR 1-10, ligands of murineTLR 1-13, ISS-1018, IC31, Imidazoquinolines, Ampligen, Ribi529, IMOxine,IRIVs, VLPs, cholera toxin, heat-labile toxin, Pam3Cys, Flagellin, GPIanchor, LNFPIII/Lewis X, antimicrobial peptides, UC-1V150, RSV fusionprotein, cdiGMP; and adjuvants suitable as antagonists including CGRPneuropeptide.

Particularly preferred, an adjuvant may be selected from adjuvants,which support induction of a Th1-immune response or maturation of naïveT-cells, such as GM-CSF, IL-12, IFNγ, any immunostimulatory nucleic acidas defined above, preferably an immunostimulatory RNA, CpG DNA, etc.

In a further preferred embodiment it is also possible that the inventivecomposition contains besides the antigen-providing nucleic acid asdisclosed herein further components which are selected from the groupcomprising: further antigens (e.g. in the form of a peptide or protein)or further antigen-encoding nucleic acids; a further immunotherapeuticagent; one or more auxiliary substances; or any further compound, whichis known to be immune stimulating due to its binding affinity (asligands) to human Toll-like receptors; and/or an adjuvant nucleic acid,preferably an immunostimulatory RNA (isRNA).

The composition of the present invention can additionally contain one ormore auxiliary substances in order to increase its immunogenicity orimmunostimulatory capacity, if desired. A synergistic action of thenucleic acid or preferably the mRNA as defined herein and of anauxiliary substance, which may be optionally contained in the inventivecomposition, is preferably achieved thereby. Depending on the varioustypes of auxiliary substances, various mechanisms can come intoconsideration in this respect. For example, compounds that permit thematuration of dendritic cells (DCs), for example lipopolysaccharides,TNF-alpha or CD40 ligand, form a first class of suitable auxiliarysubstances. In general, it is possible to use as auxiliary substance anyagent that influences the immune system in the manner of a “dangersignal” (LPS, GP96, etc.) or cytokines, such as GM-CFS, which allow animmune response to be enhanced and/or influenced in a targeted manner.Particularly preferred auxiliary substances are cytokines, such asmonokines, lymphokines, interleukins or chemokines, that further promotethe innate immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17,IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27,IL-28, IL-29, IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta,IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors,such as hGH.

Suitable adjuvants may also be selected from cationic or polycationiccompounds wherein the adjuvant is preferably prepared upon complexingthe mRNA of the composition according to the invention with the cationicor polycationic compound. Associating or complexing the mRNA of thecomposition with cationic or polycationic compounds as defined hereinpreferably provides adjuvant properties and confers a stabilizing effectto the mRNA of the composition. In particular, such preferred cationicor polycationic compounds are selected from cationic or polycationicpeptides or proteins, including protamine, nucleoline, spermin orspermidine, or other cationic peptides or proteins, such aspoly-L-lysine (PLL), poly-arginine, basic polypeptides, cell penetratingpeptides (CPPs), including HIV-binding peptides, Tat, HIV-1 Tat (HIV),Tat-derived peptides, Penetratin, VP22 derived or analog peptides, HSVVP22 (Herpes simplex), MAP, KALA or protein transduction domains (PTDs,PpT620, prolin-rich peptides, arginine-rich peptides, lysine-richpeptides, MPG-peptide(s), Pep-1, L-oligomers, Calcitonin peptide(s),Antennapedia-derived peptides (particularly from Drosophilaantennapedia), pAntp, plsl, FGF, Lactoferrin, Transportan, Buforin-2,Bac715-24, SynB, SynB(1), pVEC, hCT-derived peptides, SAP, protamine,spermine, spermidine, or histones. Further preferred cationic orpolycationic compounds may include cationic polysaccharides, for examplechitosan, polybrene, cationic polymers, e.g. polyethyleneimine (PEI),cationic lipids, e.g. DOTMA:[1-(2,3-sioleyloxy)propyl)]-N,N,N-trimethylammonium chloride, DMRIE,di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC, DODAP, DOPE:Dioleyl phosphatidylethanol-amine, DOSPA, DODAB, DOIC, DMEPC, DOGS:Dioctadecylamidoglicylspermin, DIMRI: Dimyristo-oxypropyl dimethylhydroxyethyl ammonium bromide, DOTAP:dioleoyloxy-3-(trimethylammonio)propane, DC-6-14:O,O-ditetradecanoyl-N-(α-trimethylammonioacetyl)diethanolamine chloride,CLIP1: rac-[(2,3-dioctadecyloxypropyl)(2-hydroxyethyl)]-dimethylammoniumchloride, CLIP6:rac-[2(2,3-dihexadecyloxypropyl-oxymethyloxy)ethyl]-trimethylammonium,CLIP9:rac-[2(2,3-dihexadecyloxypropyl-oxysuccinyloxy)ethyl]-trimethylammonium,oligofectamine, or cationic or polycationic polymers, e.g. modifiedpolyaminoacids, such as β-aminoacid-polymers or reversed polyamides,etc., modified polyethylenes, such as PVP(poly(N-ethyl-4-vinylpyridinium bromide)), etc., modified acrylates,such as pDMAEMA (poly(dimethylaminoethyl methylacrylate)), etc.,modified Amidoamines such as pAMAM (poly(amidoamine)), etc., modifiedpolybetaaminoester (PBAE), such as diamine end modified 1,4 butanedioldiacrylate-co-5-amino-1-pentanol polymers, etc., dendrimers, such aspolypropylamine dendrimers or pAMAM based dendrimers, etc.,polyimine(s), such as PEI: poly(ethyleneimine), poly(propyleneimine),etc., polyallylamine, sugar backbone based polymers, such ascyclodextrin based polymers, dextran based polymers, Chitosan, etc.,silan backbone based polymers, such as PMOXA-PDMS copolymers, etc.,blockpolymers consisting of a combination of one or more cationic blocks(e.g. selected of a cationic polymer as mentioned above) and of one ormore hydrophilic- or hydrophobic blocks (e.g polyethyleneglycole); etc.

Additionally, preferred cationic or polycationic proteins or peptides,which can be used as an adjuvant by complexing the mRNA of thecomposition according to the invention, may be selected from followingproteins or peptides having the following total formula (III): (Arg)I;(Lys)m; (His)n; (Orn)o; (Xaa)x, wherein l+m+n+o+x=8-15, and l, m, n or oindependently of each other may be any number selected from 0, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, provided that the overallcontent of Arg, Lys, His and Orn represents at least 50% of all aminoacids of the oligopeptide; and Xaa may be any amino acid selected fromnative (=naturally occurring) or non-native amino acids except of Arg,Lys, His or Orn; and x may be any number selected from 0, 1, 2, 3 or 4,provided, that the overall content of Xaa does not exceed 50% of allamino acids of the oligopeptide. Particularly preferred oligoargininesin this context are e.g. Arg7, Arg8, Arg9, Arg7, H3R9, R9H3, H3R9H3,YSSR9SSY, (RKH)4, Y(RKH)2R, etc.

The ratio of the nucleic acid, particularly of mRNA to the cationic orpolycationic compound in the adjuvant component may be calculated on thebasis of the nitrogen/phosphate ratio (N/P-ratio) of the entire mRNAcomplex, i.e. the ratio of positively charged (nitrogen) atoms of thecationic or polycationic compound to the negatively charged phosphateatoms of the nucleic acids. For example, 1 μg of RNA typically containsabout 3 nmol phosphate residues, provided the RNA exhibits a statisticaldistribution of bases. Additionally, 1 μg of peptide typically containsabout x nmol nitrogen residues, dependent on the molecular weight andthe number of basic amino acids. When exemplarily calculated for (Arg)9(molecular weight 1424 g/mol, 9 nitrogen atoms), 1 μg (Arg)9 containsabout 700 pmol (Arg)9 and thus 700×9=6300 pmol basic amino acids=6.3nmol nitrogen atoms. For a mass ratio of about 1:1 RNA/(Arg)9 an N/Pratio of about 2 can be calculated. When exemplarily calculated forprotamine (molecular weight about 4250 g/mol, 21 nitrogen atoms, whenprotamine from salmon is used) with a mass ratio of about 2:1 with 2 μgRNA, 6 nmol phosphate are to be calculated for the RNA; 1 μg protaminecontains about 235 pmol protamine molecules and thus 235×21=4935 pmolbasic nitrogen atoms=4.9 nmol nitrogen atoms. For a mass ratio of about2:1 RNA/protamine an N/P ratio of about 0.81 can be calculated. For amass ratio of about 8:1 RNA/protamine an N/P ratio of about 0.2 can becalculated. In the context of the present invention, an N/P-ratio ispreferably in the range of about 0.1-10, preferably in a range of about0.3-4 and most preferably in a range of about 0.5-2 or 0.7-2 regardingthe ratio of RNA:peptide in the complex, and most preferably in therange of about 0.7-1.5.

In a preferred embodiment, the composition of the present invention isobtained in two separate steps in order to obtain both, an efficientimmunostimulatory effect and efficient translation of the nucleic acid,particularly the mRNA according to the invention. Therein, a so called“adjuvant component” is prepared by complexing—in a first step—an mRNAas defined herein of the adjuvant component with a cationic orpolycationic compound in a specific ratio to form a stable complex. Inthis context, it is important, that no free cationic or polycationiccompound or only a negligibly small amount remains in the adjuvantcomponent after complexing the mRNA. Accordingly, the ratio of the mRNAand the cationic or polycationic compound in the adjuvant component istypically selected in a range that the mRNA is entirely complexed and nofree cationic or polycationic compound or only a negligible small amountremains in the composition. Preferably the ratio of the adjuvantcomponent, i.e. the ratio of the mRNA to the cationic or polycationiccompound is selected from a range of about 6:1 (w/w) to about 0.25:1(w/w), more preferably from about 5:1 (w/w) to about 0.5:1 (w/w), evenmore preferably of about 4:1 (w/w) to about 1:1 (w/w) or of about 3:1(w/w) to about 1:1 (w/w), and most preferably a ratio of about 3:1 (w/w)to about 2:1 (w/w).

According to a preferred embodiment, the nucleic acid, particularly themRNA of the invention comprising at least one mRNA sequence comprisingat least one coding region as defined herein is added in a second stepto the complexed mRNA of the adjuvant component in order to form the(immunostimulatory) composition of the invention. Therein, the mRNA ofthe composition according to the invention is added as free mRNA, whichis not complexed by other compounds. Prior to addition, the free mRNA isnot complexed and will preferably not undergo any detectable orsignificant complexation reaction upon the addition of the adjuvantcomponent. This is due to the strong binding of the cationic orpolycationic compound to the above described mRNA according to theinvention comprised in the adjuvant component. In other words, when themRNA comprising at least one coding region as defined herein is added tothe “adjuvant component”, preferably no free or substantially no freecationic or polycationic compound is present, which could form a complexwith the free mRNA. Accordingly, an efficient translation of the mRNA ofthe composition is possible in vivo. Therein, the free mRNA, may occuras a mono-, di-, or multicistronic mRNA, i.e. an mRNA which carries thecoding sequences of one or more proteins. Such coding sequences in di-,or even multicistronic mRNA may be separated by at least one IRESsequence, e.g. as defined herein.

In a particularly preferred embodiment, the free nucleic acid,particularly the mRNA as defined herein, which is comprised in thecomposition of the present invention, may be identical or different tothe RNA as defined herein, which is comprised in the adjuvant componentof the composition, depending on the specific requirements of therapy.Even more preferably, the free RNA, which is comprised in thecomposition according to the invention, is identical to the RNA of theadjuvant component of the inventive composition.

In a particularly preferred embodiment, the composition according to theinvention comprises the nucleic acid, particularly the mRNA of theinvention, which encodes at least one NIPAH virus antigenic peptide orprotein as defined herein and wherein said mRNA is present in thecomposition partially as free mRNA and partially as complexed mRNA.Preferably, the mRNA as defined herein is complexed as described aboveand the same mRNA is then added as free mRNA, wherein preferably thecompound, which is used for complexing the mRNA is not present in freeform in the composition at the moment of addition of the free mRNAcomponent.

The ratio of the first component (i.e. the adjuvant component comprisingor consisting of the nucleic acid, particularly the mRNA as definedherein complexed with a cationic or polycationic compound) and thesecond component (i.e. the free mRNA as defined herein) may be selectedin the inventive composition according to the specific requirements of aparticular therapy. Typically, the ratio of the mRNA in the adjuvantcomponent and the at least one free mRNA (mRNA in the adjuvantcomponent:free mRNA) of the composition according to the invention isselected such that a significant stimulation of the innate immune systemis elicited due to the adjuvant component. In parallel, the ratio isselected such that a significant amount of the free mRNA can be providedin vivo leading to an efficient translation and concentration of theexpressed protein in vivo, e.g. the at least one NIPAH virus antigenicpeptide or protein as defined herein. Preferably the ratio of the mRNAin the adjuvant component:free mRNA in the inventive composition isselected from a range of about 5:1 (w/w) to about 1:10 (w/w), morepreferably from a range of about 4:1 (w/w) to about 1:8 (w/w), even morepreferably from a range of about 3:1 (w/w) to about 1:5 (w/w) or 1:3(w/w), and most preferably the ratio of mRNA in the adjuvantcomponent:free mRNA in the inventive composition is selected from aratio of about 1:1 (w/w).

Additionally or alternatively, the ratio of the first component (i.e.the adjuvant component comprising or consisting of the nucleic acid,particularly the mRNA complexed with a cationic or polycationiccompound) and the second component (i.e. the free mRNA) may becalculated on the basis of the nitrogen/phosphate ratio (N/P-ratio) ofthe entire mRNA complex. In the context of the present invention, anN/P-ratio is preferably in the range of about 0.1-10, preferably in arange of about 0.3-4 and most preferably in a range of about 0.5-2 or0.7-2 regarding the ratio of mRNA:peptide in the complex, and mostpreferably in the range of about 0.7-1.5.

Additionally or alternatively, the ratio of the first component (i.e.the adjuvant component comprising or consisting of the nucleic acid,particularly the mRNA complexed with a cationic or polycationiccompound) and the second component (i.e. the free mRNA) may also beselected in the composition according to the invention on the basis ofthe molar ratio of both mRNAs to each other, i.e. the mRNA of theadjuvant component, being complexed with a cationic or polycationiccompound and the free mRNA of the second component. Typically, the molarratio of the mRNA of the adjuvant component to the free mRNA of thesecond component may be selected such, that the molar ratio suffices theabove (w/w) and/or N/P-definitions. More preferably, the molar ratio ofthe mRNA of the adjuvant component to the free mRNA of the secondcomponent may be selected e.g. from a molar ratio of about 0.001:1,0.01:1, 0.1:1, 0.2:1, 0.3:1, 0.4:1, 0.5:1, 0.6:1, 0.7:1, 0.8:1, 0.9:1,1:1, 1:0.9, 1:0.8, 1:0.7, 1:0.6, 1:0.5, 1:0.4, 1:0.3, 1:0.2, 1:0.1,1:0.01, 1:0.001, etc. or from any range formed by any two of the abovevalues, e.g. a range selected from about 0.001:1 to 1:0.001, including arange of about 0.01:1 to 1:0.001, 0.1:1 to 1:0.001, 0.2:1 to 1:0.001,0.3:1 to 1:0.001, 0.4:1 to 1:0.001, 0.5:1 to 1:0.001, 0.6:1 to 1:0.001,0.7:1 to 1:0.001, 0.8:1 to 1:0.001, 0.9:1 to 1:0.001, 1:1 to 1:0.001,1:0.9 to 1:0.001, 1:0.8 to 1:0.001, 1:0.7 to 1:0.001, 1:0.6 to 1:0.001,1:0.5 to 1:0.001, 1:0.4 to 1:0.001, 1:0.3 to 1:0.001, 1:0.2 to 1:0.001,1:0.1 to 1:0.001, 1:0.01 to 1:0.001, or a range of about 0.01:1 to1:0.01, 0.1:1 to 1:0.01, 0.2:1 to 1:0.01, 0.3:1 to 1:0.01, 0.4:1 to1:0.01, 0.5:1 to 1:0.01, 0.6:1 to 1:0.01, 0.7:1 to 1:0.01, 0.8:1 to1:0.01, 0.9:1 to 1:0.01, 1:1 to 1:0.01, 1:0.9 to 1:0.01, 1:0.8 to1:0.01, 1:0.7 to 1:0.01, 1:0.6 to 1:0.01, 1:0.5 to 1:0.01, 1:0.4 to1:0.01, 1:0.3 to 1:0.01, 1:0.2 to 1:0.01, 1:0.1 to 1:0.01, 1:0.01 to1:0.01, or including a range of about 0.001:1 to 1:0.01, 0.001:1 to1:0.1, 0.001:1 to 1:0.2, 0.001:1 to 1:0.3, 0.001:1 to 1:0.4, 0.001:1 to1:0.5, 0.001:1 to 1:0.6, 0.001:1 to 1:0.7, 0.001:1 to 1:0.8, 0.001:1 to1:0.9, 0.001:1 to 1:1, 0.001 to 0.9:1, 0.001 to 0.8:1, 0.001 to 0.7:1,0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to 0.4:1, 0.001 to 0.3:1, 0.001 to0.2:1, 0.001 to 0.1:1, or a range of about 0.01:1 to 1:0.01, 0.01:1 to1:0.1, 0.01:1 to 1:0.2, 0.01:1 to 1:0.3, 0.01:1 to 1:0.4, 0.01:1 to1:0.5, 0.01:1 to 1:0.6, 0.01:1 to 1:0.7, 0.01:1 to 1:0.8, 0.01:1 to1:0.9, 0.01:1 to 1:1, 0.001 to 0.9:1, 0.001 to 0.8:1, 0.001 to 0.7:1,0.001 to 0.6:1, 0.001 to 0.5:1, 0.001 to 0.4:1, 0.001 to 0.3:1, 0.001 to0.2:1, 0.001 to 0.1:1, etc.

Even more preferably, the molar ratio of the nucleic acid, particularlythe mRNA of the adjuvant component to the free mRNA of the secondcomponent may be selected e.g. from a range of about 0.01:1 to 1:0.01.Most preferably, the molar ratio of the mRNA of the adjuvant componentto the free mRNA of the second component may be selected e.g. from amolar ratio of about 1:1. Any of the above definitions with regard to(w/w) and/or N/P ratio may also apply.

Suitable adjuvants may furthermore be selected from nucleic acids havingthe formula (Va): GlXmGn, wherein: G is guanosine, uracil or an analogueof guanosine or uracil; X is guanosine, uracil, adenosine, thymidine,cytosine or an analogue of the above-mentioned nucleotides; l is aninteger from 1 to 40, wherein when l=1 G is guanosine or an analoguethereof, when l>1 at least 50% of the nucleotides are guanosine or ananalogue thereof; m is an integer and is at least 3; wherein when m=3 Xis uracil or an analogue thereof, when m>3 at least 3 successive uracilsor analogues of uracil occur; n is an integer from 1 to 40, wherein whenn=1 G is guanosine or an analogue thereof, when n>1 at least 50% of thenucleotides are guanosine or an analogue thereof, or formula (Vb):(NuGlXmGnNv)a, wherein: G is guanosine (guanine), uridine (uracil) or ananalogue of guanosine (guanine) or uridine (uracil), preferablyguanosine (guanine) or an analogue thereof; X is guanosine (guanine),uridine (uracil), adenosine (adenine), thymidine (thymine), cytidine(cytosine), or an analogue of these nucleotides (nucleosides),preferably uridine (uracil) or an analogue thereof; N is a nucleic acidsequence having a length of about 4 to 50, preferably of about 4 to 40,more preferably of about 4 to 30 or 4 to 20 nucleic acids, each Nindependently being selected from guanosine (guanine), uridine (uracil),adenosine (adenine), thymidine (thymine), cytidine (cytosine) or ananalogue of these nucleotides (nucleosides); a is an integer from 1 to20, preferably from 1 to 15, most preferably from 1 to 10; l is aninteger from 1 to 40, wherein when l=1, G is guanosine (guanine) or ananalogue thereof, when l>1, at least 50% of these nucleotides(nucleosides) are guanosine (guanine) or an analogue thereof; m is aninteger and is at least 3; wherein when m=3, X is uridine (uracil) or ananalogue thereof, and when m>3, at least 3 successive uridines (uracils)or analogues of uridine (uracil) occur; n is an integer from 1 to 40,wherein when n=1, G is guanosine (guanine) or an analogue thereof, whenn>1, at least 50% of these nucleotides (nucleosides) are guanosine(guanine) or an analogue thereof; u,v may be independently from eachother an integer from 0 to 50, preferably wherein when u=0, v≥1, or whenv=0, u≥1; wherein the nucleic acid molecule of formula (Vb) has a lengthof at least 50 nucleotides, preferably of at least 100 nucleotides, morepreferably of at least 150 nucleotides, even more preferably of at least200 nucleotides and most preferably of at least 250 nucleotides.

Other suitable adjuvants may furthermore be selected from nucleic acidshaving the formula (VI): CIXmCn, wherein: C is cytosine, uracil or ananalogue of cytosine or uracil; X is guanosine, uracil, adenosine,thymidine, cytosine or an analogue of the above-mentioned nucleotides; lis an integer from 1 to 40, wherein when l=1 C is cytosine or ananalogue thereof, when l>1 at least 50% of the nucleotides are cytosineor an analogue thereof; m is an integer and is at least 3; wherein whenm=3 X is uracil or an analogue thereof, when m>3 at least 3 successiveuracils or analogues of uracil occur; n is an integer from 1 to 40,wherein when n=1 C is cytosine or an analogue thereof, when n>1 at least50% of the nucleotides are cytosine or an analogue thereof.

In this context the disclosure of WO2008014979 and WO2009095226 is alsoincorporated herein by reference.

Vaccine:

In a further aspect, the present invention provides a vaccine, which isbased on the nucleic acid, particularly the mRNA sequence according tothe invention comprising at least one coding region as defined herein.The vaccine according to the invention is preferably a (pharmaceutical)composition as defined herein.

The vaccine according to the invention is based on the same componentsas the (pharmaceutical) composition described herein. Insofar, it may bereferred to the description of the (pharmaceutical) composition asprovided herein. Preferably, the vaccine according to the inventioncomprises at least one nucleic acid comprising at least one nucleic acidsequence as defined herein and a pharmaceutically acceptable carrier. Inembodiments, where the vaccine comprises more than one nucleic acid,particularly more than one mRNA sequence (such as a plurality of RNAsequences according to the invention, wherein each preferably encodes adistinct antigenic peptide or protein), the vaccine may be provided inphysically separate form and may be administered by separateadministration steps. The vaccine according to the invention maycorrespond to the (pharmaceutical) composition as described herein,especially where the mRNA sequences are provided by one singlecomposition. However, the inventive vaccine may also be providedphysically separated. For instance, in embodiments, wherein the vaccinecomprises more than one mRNA sequences/species, these RNA species may beprovided such that, for example, two, three, four, five or six separatecompositions, which may contain at least one mRNA species/sequence each(e.g, three distinct mRNA species/sequences), each encoding distinctantigenic peptides or proteins, are provided, which may or may not becombined. Also, the inventive vaccine may be a combination of at leasttwo distinct compositions, each composition comprising at least one mRNAencoding at least one of the antigenic peptides or proteins definedherein. Alternatively, the vaccine may be provided as a combination ofat least one mRNA, preferably at least two, three, four, five, six ormore mRNAs, each encoding one of the antigenic peptides or proteinsdefined herein. The vaccine may be combined to provide one singlecomposition prior to its use or it may be used such that more than oneadministration is required to administer the distinct mRNAsequences/species encoding any of the antigenic peptides or proteins asdefined herein. If the vaccine contains at least one mRNA sequence,typically at least two mRNA sequences, encoding the antigen combinationsdefined herein, it may e.g. be administered by one single administration(combining all mRNA species/sequences), by at least two separateadministrations. Accordingly, any combination of mono-, bi- ormulticistronic mRNAs encoding the at least one antigenic peptide orprotein or any combination of antigens as defined herein (and optionallyfurther antigens), provided as separate entities (containing one mRNAspecies) or as combined entity (containing more than one mRNA species),is understood as a vaccine according to the present invention.

As with the (pharmaceutical) composition according to the presentinvention, the entities of the vaccine may be provided in liquid and orin dry (e.g. lyophilized) form. They may contain further components, inparticular further components allowing for its pharmaceutical use. Thevaccine or the (pharmaceutical) composition may, e.g., additionallycontain a pharmaceutically acceptable carrier and/or further auxiliarysubstances and additives and/or adjuvants.

The vaccine or (pharmaceutical) composition typically comprises a safeand effective amount of the nucleic acid, particularly mRNA according tothe invention as defined herein, encoding an antigenic peptide orprotein as defined herein or a fragment or variant thereof or acombination of antigens, preferably as defined herein. As used herein,“safe and effective amount” means an amount of the mRNA that issufficient to significantly induce a positive modification of cancer ora disease or disorder related to cancer. At the same time, however, a“safe and effective amount” is small enough to avoid seriousside-effects, that is to say to permit a sensible relationship betweenadvantage and risk. The determination of these limits typically lieswithin the scope of sensible medical judgment. In relation to thevaccine or (pharmaceutical) composition of the present invention, theexpression “safe and effective amount” preferably means an amount of themRNA (and thus of the encoded virus antigen) that is suitable forstimulating the adaptive immune system in such a manner that noexcessive or damaging immune reactions are achieved but, preferably,also no such immune reactions below a measurable level. Such a “safe andeffective amount” of the mRNA of the (pharmaceutical) composition orvaccine as defined herein may furthermore be selected in dependence ofthe type of mRNA, e.g. monocistronic, bi- or even multicistronic mRNA,since a bi- or even multicistronic mRNA may lead to a significantlyhigher expression of the encoded virus antigen(s) than the use of anequal amount of a monocistronic mRNA. A “safe and effective amount” ofthe mRNA of the (pharmaceutical) composition or vaccine as defined abovewill furthermore vary in connection with the particular condition to betreated and also with the age and physical condition of the patient tobe treated, the severity of the condition, the duration of thetreatment, the nature of the accompanying therapy, of the particularpharmaceutically acceptable carrier used, and similar factors, withinthe knowledge and experience of the accompanying doctor. The vaccine orcomposition according to the invention can be used according to theinvention for human and also for veterinary medical purposes (mammals,vertebrates), as a pharmaceutical composition or as a vaccine.

In a preferred embodiment, the artificial nucleic acid, vaccine orcomposition according to the invention is used as pharmaceuticalcomposition or as a vaccine in the prophylaxis or treatment of disordersrelated to Henipavirus and/or Nipah virus and/or Hendra virus inmammals, wherein the mammal may be selected from canines (e.g., dogs),felines (e.g., cats), equines (e.g., horses), bovines (e.g., cattle)porcine (e.g., pigs), as well as bats, flying foxes, rodents etc.

In a preferred embodiment, the nucleic acid, particularly the mRNA ofthe (pharmaceutical) composition, vaccine or kit of parts according tothe invention is provided in lyophilized form. Preferably, thelyophilized mRNA is reconstituted in a suitable buffer, advantageouslybased on an aqueous carrier, prior to administration, e.g.Ringer-Lactate solution, which is preferred, Ringer solution, aphosphate buffer solution. In a preferred embodiment, the(pharmaceutical) composition, the vaccine or the kit of parts accordingto the invention contains at least one, two, three, four, five, six ormore mRNAs, preferably mRNAs which are provided separately inlyophilized form (optionally together with at least one furtheradditive) and which are preferably reconstituted separately in asuitable buffer (such as Ringer-Lactate solution) prior to their use soas to allow individual administration of each of the (monocistronic)mRNAs.

The vaccine or (pharmaceutical) composition according to the inventionmay typically contain a pharmaceutically acceptable carrier. Theexpression “pharmaceutically acceptable carrier” as used hereinpreferably includes the liquid or non-liquid basis of the inventivevaccine. If the inventive vaccine is provided in liquid form, thecarrier will be water, typically pyrogen-free water; isotonic saline orbuffered (aqueous) solutions, e.g phosphate, citrate etc. bufferedsolutions. Particularly for injection of the inventive vaccine, water orpreferably a buffer, more preferably an aqueous buffer, may be used,containing a sodium salt, preferably at least 50 mM of a sodium salt, acalcium salt, preferably at least 0.01 mM of a calcium salt, andoptionally a potassium salt, preferably at least 3 mM of a potassiumsalt. According to a preferred embodiment, the sodium, calcium and,optionally, potassium salts may occur in the form of their halogenides,e.g. chlorides, iodides, or bromides, in the form of their hydroxides,carbonates, hydrogen carbonates, or sulfates, etc. Without being limitedthereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃,NaHCO₃, Na₂SO₄, examples of the optional potassium salts include e.g.KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts includee.g. CaCl₂), Cal₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Furthermore, organicanions of the aforementioned cations may be contained in the buffer.According to a more preferred embodiment, the buffer suitable forinjection purposes as defined above, may contain salts selected fromsodium chloride (NaCl), calcium chloride (CaCl₂)) and optionallypotassium chloride (KCl), wherein further anions may be presentadditional to the chlorides. CaCl₂ can also be replaced by another saltlike KCl. Typically, the salts in the injection buffer are present in aconcentration of at least 50 mM sodium chloride (NaCl), at least 3 mMpotassium chloride (KCl) and at least 0.01 mM calcium chloride (CaCl₂)).The injection buffer may be hypertonic, isotonic or hypotonic withreference to the specific reference medium, i.e. the buffer may have ahigher, identical or lower salt content with reference to the specificreference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due toosmosis or other concentration effects. Reference media are e.g. in “invivo” methods occurring liquids such as blood, lymph, cytosolic liquids,or other body liquids, or e.g. liquids, which may be used as referencemedia in “in vitro” methods, such as common buffers or liquids. Suchcommon buffers or liquids are known to a skilled person. Ringer-Lactatesolution is particularly preferred as a liquid basis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well, which are suitable foradministration to a person. The term “compatible” as used herein meansthat the constituents of the inventive vaccine are capable of beingmixed with the nucleic acid, particularly the mRNA according to theinvention as defined herein, in such a manner that no interactionoccurs, which would substantially reduce the pharmaceuticaleffectiveness of the inventive vaccine under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a person to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose, trehalose and sucrose; starches, such as, forexample, corn starch or potato starch; dextrose; cellulose and itsderivatives, such as, for example, sodium carboxymethylcellulose,ethylcellulose, cellulose acetate; powdered tragacanth; malt; gelatin;tallow; solid glidants, such as, for example, stearic acid, magnesiumstearate; calcium sulfate; vegetable oils, such as, for example,groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oilfrom theobroma; polyols, such as, for example, polypropylene glycol,glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.

The choice of a pharmaceutically acceptable carrier is determined, inprinciple, by the manner, in which the pharmaceutical composition orvaccine according to the invention is administered. The composition orvaccine can be administered, for example, systemically or locally.Routes for systemic administration in general include, for example,transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,composition or vaccines according to the present invention may beadministered by an intradermal, subcutaneous, or intramuscular route,preferably by injection, which may be needle-free and/or needleinjection. Compositions/vaccines are therefore preferably formulated inliquid or solid form. The suitable amount of the vaccine or compositionaccording to the invention to be administered can be determined byroutine experiments, e.g. by using animal models. Such models include,without implying any limitation, rabbit, sheep, mouse, rat, dog andnon-human primate models. Preferred unit dose forms for injectioninclude sterile solutions of water, physiological saline or mixturesthereof. The pH of such solutions should be adjusted to about 7.4.Suitable carriers for injection include hydrogels, devices forcontrolled or delayed release, polylactic acid and collagen matrices.Suitable pharmaceutically acceptable carriers for topical applicationinclude those which are suitable for use in lotions, creams, gels andthe like. If the inventive composition or vaccine is to be administeredperorally, tablets, capsules and the like are the preferred unit doseform. The pharmaceutically acceptable carriers for the preparation ofunit dose forms which can be used for oral administration are well knownin the prior art. The choice thereof will depend on secondaryconsiderations such as taste, costs and storability, which are notcritical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

The inventive vaccine or composition can additionally contain one ormore auxiliary substances in order to further increase theimmunogenicity. A synergistic action of the nucleic acid contained inthe inventive composition and of an auxiliary substance, which may beoptionally be co-formulated (or separately formulated) with theinventive vaccine or composition as described above, is preferablyachieved thereby. Depending on the various types of auxiliarysubstances, various mechanisms may play a role in this respect. Forexample, compounds that permit the maturation of dendritic cells (DCs),for example lipopolysaccharides, TNF-alpha or CD40 ligand, form a firstclass of suitable auxiliary substances. In general, it is possible touse as auxiliary substance any agent that influences the immune systemin the manner of a “danger signal” (LPS, GP96, etc.) or cytokines, suchas GM-CFS, which allow an immune response produced by theimmune-stimulating adjuvant according to the invention to be enhancedand/or influenced in a targeted manner. Particularly preferred auxiliarysubstances are cytokines, such as monokines, lymphokines, interleukinsor chemokines, that—additional to induction of the adaptive immuneresponse by the encoded at least one antigen—promote the innate immuneresponse, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20,IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30,IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF,M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH. Preferably,such immunogenicity increasing agents or compounds are providedseparately (not co-formulated with the inventive vaccine or composition)and administered individually.

Further additives which may be included in the inventive vaccine orcomposition are emulsifiers, such as, for example, Tween; wettingagents, such as, for example, sodium lauryl sulfate; colouring agents;taste-imparting agents, pharmaceutical carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

The inventive vaccine or composition can also additionally contain anyfurther compound, which is known to be immune-stimulating due to itsbinding affinity (as ligands) to human Toll-like receptors TLR1, TLR2,TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or due to its bindingaffinity (as ligands) to murine Toll-like receptors TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventive vaccineor composition in this context, may be CpG nucleic acids, in particularCpG-RNA or CpG-DNA. A CpG-RNA or CpG-DNA can be a single-strandedCpG-DNA (ss CpG-DNA), a double-stranded CpG-DNA (dsDNA), asingle-stranded CpG-RNA (ss CpG-RNA) or a double-stranded CpG-RNA (dsCpG-RNA). The CpG nucleic acid is preferably in the form of CpG-RNA,more preferably in the form of single-stranded CpG-RNA (ss CpG-RNA). TheCpG nucleic acid preferably contains at least one or more (mitogenic)cytosine/guanine dinucleotide sequence(s) (CpG motif(s)). According to afirst preferred alternative, at least one CpG motif contained in thesesequences, that is to say the C (cytosine) and the G (guanine) of theCpG motif, is unmethylated. All further cytosines or guanines optionallycontained in these sequences can be either methylated or unmethylated.According to a further preferred alternative, however, the C (cytosine)and the G (guanine) of the CpG motif can also be present in methylatedform.

In a further aspect, the present invention concerns a polypeptideencoded by the inventive artificial nucleic acid as described herein, ora fragment of said polypeptide.

In a further aspect, the present invention provides a compositioncomprising at least one of the inventive polypeptides as describedherein. In a preferred embodiment, the inventive composition comprisesone type of polypeptide as described herein. Alternatively, theinventive composition may comprise at least two different inventivepolypeptides as described herein.

In a preferred embodiment, the at least one of the inventivepolypeptides comprises at least one protein or peptides according to SEQID NOs: 1-7, 12-18, 573-579, 584-590, 807-813, 818-824, 1041-1047,1052-1058, 1513-1515 a fragment or variant thereof.

Preferably, the inventive composition comprises or consists of at leastone of the inventive polypeptides described herein and apharmaceutically acceptable carrier. In this context, thepharmaceutically acceptable carrier as well as optional furthercomponents of the composition preferably as described herein, withrespect to the inventive composition, comprises at least one inventiveartificial nucleic acid.

In another embodiment, the inventive composition comprises or consistsof at least one of the inventive polypeptides described herein and apharmaceutically acceptable carrier and at least one adjuvant.

In another embodiment, the inventive composition comprises or consistsof at least one of the inventive polypeptides described herein and apharmaceutically acceptable carrier and at least one adjuvant and atleast one inventive nucleic acid as defined herein.

In a further aspect, the invention concerns a vaccine comprising theinventive composition comprising at least one of the polypeptidesaccording to the invention. Therein, the at least one of the inventivepolypeptides preferably elicits an adaptive immune response uponadministration to a subject. More preferably, the vaccine according tothe invention comprising at least one of the inventive polypeptides orthe inventive composition comprising at least one of the polypeptidesaccording to the invention is preferably a vaccine as described herein.Reference is made to the respective description herein.

As used herein, the term “inventive composition” may refer to theinventive composition comprising at least one artificial nucleic acidaccording to the invention. Likewise, the term “inventive vaccine”, asused in this context, may refer to an inventive vaccine, which is basedon the inventive artificial nucleic acid, i.e. which comprises at leastone artificial nucleic acid according to the invention or whichcomprises the inventive composition comprising said artificial nucleicacid.

Kit:

According to another embodiment, the present invention also provideskits, particularly kits of parts, comprising the artificial nucleic acidaccording to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide or the inventive vaccineas described herein, optionally a liquid vehicle for solubilising andoptionally technical instructions with information on the administrationand dosage of the artificial nucleic acid according as described herein,the inventive composition comprising at least one artificial nucleicacid according to the invention, the inventive polypeptides as describedherein, the inventive composition comprising at least one inventivepolypeptide or the inventive vaccine. The technical instructions maycontain information about administration and dosage. Such kits,preferably kits of parts, may be applied e.g. for any of theapplications or uses mentioned herein, preferably for the use of theartificial nucleic acid according as described herein, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, the inventive polypeptides as described herein, theinventive composition comprising at least one inventive polypeptide orthe inventive vaccine for the treatment or prophylaxis of a Henipvirusand/or a Nipah virus and/or a Hendra virus infection or diseases ordisorders related thereto. The kits may also be applied for the use ofthe artificial nucleic acid according as described herein, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, the inventive polypeptides as described herein, theinventive composition comprising at least one inventive polypeptide orthe inventive vaccine for the treatment or prophylaxis of Henipvirusand/or a Nipah virus and/or a Hendra virus infection or diseases ordisorders related thereto, wherein the artificial nucleic acid accordingas described herein, the inventive composition comprising at least oneartificial nucleic acid according to the invention, the inventivepolypeptides as described herein, the inventive composition comprisingat least one inventive polypeptide or the inventive vaccine may induceor enhance an immune response in a mammal as defined above. Preferably,the artificial nucleic acid according as described herein, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, or the inventive vaccine is provided in a separate partof the kit, wherein the artificial nucleic acid according as describedherein, the inventive composition comprising at least one artificialnucleic acid according to the invention, or the inventive vaccine arepreferably lyophilised. More preferably, the kit further contains as apart a vehicle for solubilising the artificial nucleic acid according asdescribed herein, the inventive composition comprising at least oneartificial nucleic acid according to the invention, or the inventivevaccine, the vehicle preferably being Ringer-lactate solution. Any ofthe above kits may be used in a treatment or prophylaxis as definedabove. More preferably, any of the above kits may be used as a vaccine,preferably a vaccine against Henipvirus and/or a Nipah virus and/or aHendra virus infection or a related disease or disorder.

Any of the above kits may be used in a treatment or prophylaxis asdefined above. More preferably, any of the above kits may be used as avaccine, preferably a vaccine against Henipvirus and/or a Nipah virusand/or a Hendra virus infection or a related disease or disorder.

Application and Medical Use:

According to one aspect of the present invention, the nucleic sequence,the (pharmaceutical) composition or the vaccine may be used according tothe invention (for the preparation of a medicament) as a medicament.

The present invention furthermore provides several applications and usesof the artificial nucleic acid according to the invention, the inventivecomposition comprising at least one artificial nucleic acid according tothe invention, the inventive polypeptides as described herein, theinventive composition comprising at least one inventive polypeptide orthe inventive vaccine or of kits comprising same. In particular, theinventive (pharmaceutical) composition(s) or the inventive vaccine maybe used for human and also for veterinary medical purposes, preferablyfor human medical purposes, as a pharmaceutical composition in generalor as a vaccine.

In a further aspect, the invention provides the artificial nucleic acidaccording to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide, the inventive vaccine orthe inventive kit or kit of parts for use in a method of prophylactic(pre-exposure prophylaxis or post-exposure prophylaxis) and/ortherapeutic treatment of Henipvirus and/or a Nipah virus and/or a Hendravirus infections. Consequently, in a further aspect, the presentinvention is directed to the first medical use of the artificial nucleicacid according to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide, the inventive vaccine orthe inventive kit or kit of parts as defined herein as a medicament.Particularly, the invention provides the use of an artificial nucleicacid comprising at least one coding region encoding at least onepolypeptide comprising at least one Henipvirus and/or a Nipah virusand/or a Hendra virus protein or peptide as defined herein, or afragment or variant thereof as described herein for the preparation of amedicament.

According to another aspect, the present invention is directed to thesecond medical use of the artificial nucleic acid according to theinvention, the inventive composition comprising at least one artificialnucleic acid according to the invention, the inventive polypeptides asdescribed herein, the inventive composition comprising at least oneinventive polypeptide, the inventive vaccine or the inventive kit or kitof parts for the treatment of an infection with Henipvirus and/or aNipah virus and/or a Hendra virus or a disease or disorders related toan infection with Henipvirus and/or a Nipah virus and/or a Hendra virusas defined herein.

Particularly, the artificial nucleic acid comprising at least one codingregion encoding at least one polypeptide comprising at least oneantigenic protein or peptide as defined herein, or a fragment or variantthereof as described herein to be used in a method as said above is anartificial nucleic acid formulated together with a pharmaceuticallyacceptable vehicle and an optionally additional adjuvant and anoptionally additional further component as defined herein.

The invention provides the artificial nucleic acid according to theinvention, the inventive composition comprising at least one artificialnucleic acid according to the invention, the inventive polypeptides asdescribed herein, the inventive composition comprising at least oneinventive polypeptide, the inventive vaccine or the inventive kit or kitof parts for medical use, in particular for the treatment of aninfection with Henipvirus and/or a Nipah virus and/or a Hendra virus ora disease or disorders related to an infection with Henipvirus and/or aNipah virus and/or a Hendra virus, wherein preferably an infection withHenipvirus and/or a Nipah virus and/or a Hendra virus may involve anyHenipvirus and/or a Nipah virus and/or a Hendra virus as defined herein.

As used herein, “a disorder related to a Henipvirus and/or a Nipah virusand/or a Hendra virus infection” or “a disease related to a Henipvirusand/or a Nipah virus and/or a Hendra virus infection” may preferablycomprise a complication of Henipvirus and/or a Nipah virus and/or aHendra virus infection. Complications and disease related disordersassociated with Nipah virus infection include fever and headache,followed by drowsiness, disorientation and mental confusion, respiratoryillness and encephalitis (inflammation of the brain). Complications anddisease related disorders associated with Hendra virus include variousrespiratory and neurologic complications and symptoms.

In a preferred embodiment, the inventive composition or vaccine is thusused for treatment or prophylaxis, preferably prophylaxis, ofcomplications associated with a Nipah virus infection.

In a preferred embodiment, the inventive composition or vaccine is thusused for treatment or prophylaxis, preferably prophylaxis, ofcomplications associated with a Hendra virus infection.

In a preferred embodiment, the inventive composition or vaccine is thusused for treatment or prophylaxis, preferably prophylaxis, ofcomplications associated with a Henipavirus infection.

The inventive composition or the inventive vaccine, in particular theinventive composition comprising at least one artificial nucleic acidaccording to the invention, the inventive polypeptides as describedherein or the inventive composition comprising at least one inventivepolypeptide, can be administered, for example, systemically or locally.Routes for systemic administration in general include, for example,transdermal, oral, parenteral routes, including subcutaneous,intravenous, intramuscular, intraarterial, intradermal andintraperitoneal injections and/or intranasal administration routes.Routes for local administration in general include, for example, topicaladministration routes but also intradermal, transdermal, subcutaneous,or intramuscular injections or intralesional, intracranial,intrapulmonal, intracardial, and sublingual injections. More preferably,vaccines may be administered by an intradermal, subcutaneous, orintramuscular route. Inventive vaccines are therefore preferablyformulated in liquid (or sometimes in solid) form. Preferably, theinventive vaccine may be administered by conventional needle injectionor needle-free jet injection. In a preferred embodiment the inventivevaccine or composition may be administered by jet injection as definedherein, preferably intramuscularly or intradermally, more preferablyintradermally.

In a preferred embodiment, a single dose of the inventive artificialnucleic acid, composition or vaccine comprises a specific amount of theartificial nucleic acid according to the invention.

In embodiments, the inventive artificial nucleic acid is provided in anamount of 5 μg, 10 μg, 15 μg, 20 μg, 25 μg, 30 μg, 35 μg, 40 μg, 45 μg,50 μg, 60 μg, 70 μg, 80 μg, 90 μg, or 100 μg. Preferably, the inventiveartificial nucleic acid is provided in an amount of at least 5 μg perdose, preferably in an amount of from 10 to 500 μg per dose, morepreferably in an amount of from 20 to 200 μg per dose. Morespecifically, in the case of intradermal injection, which is preferablycarried out by using a conventional needle, the amount of the inventiveartificial nucleic acid comprised in a single dose is typically at least5 μg, preferably from 10 μg to 500 μg, more preferably from 20 μg to 200μg, even more preferably from 30 μg to 100 μg. In the case ofintradermal injection, which is preferably carried out via jet injection(e.g. using a Tropis device), the amount of the inventive artificialnucleic acid comprised in a single dose is typically at least 10 μg,preferably from 20 μg to 200 μg, more preferably from 30 μg to 100 μg.Moreover, in the case of intramuscular injection, which is preferablycarried out by using a conventional needle or via jet injection, theamount of the inventive artificial nucleic acid comprised in a singledose is typically at least 1 μg, preferably from 1 μg to 500 μg, morepreferably from 5 μg to 500 μg, even more preferably from 10 μg to 200μg.

The immunization protocol for the treatment or prophylaxis of aHenipvirus and/or a Nipah virus and/or a Hendra virus infection, i.e theimmunization of a subject against Henipvirus and/or a Nipah virus and/ora Hendra virus, typically comprises a series of single doses or dosagesof the inventive composition or the inventive vaccine. A single dosage,as used herein, refers to the initial/first dose, a second dose or anyfurther doses, respectively, which are preferably administered in orderto “boost” the immune reaction.

According to a preferred embodiment, the artificial nucleic acidaccording to the invention, the inventive composition comprising atleast one artificial nucleic acid according to the invention, theinventive polypeptides as described herein, the inventive compositioncomprising at least one inventive polypeptide, the inventive vaccine orthe inventive kit or kit of parts is provided for use in treatment orprophylaxis, preferably treatment or prophylaxis of a Henipvirus and/ora Nipah virus and/or a Hendra virus infection or a related disorder ordisease, wherein the treatment or prophylaxis comprises theadministration of a further active pharmaceutical ingredient. Morepreferably, in the case of the inventive vaccine or composition, whichis based on the inventive artificial nucleic acid, a polypeptide may beco-administered as a further active pharmaceutical ingredient. Forexample, at least one Henipvirus and/or a Nipah virus and/or a Hendravirus protein or peptide as described herein, or a fragment or variantthereof, may be co-administered in order to induce or enhance an immuneresponse. Likewise, in the case of the inventive vaccine or composition,which is based on the inventive polypeptide as described herein, anartificial nucleic acid as described herein may be co-administered as afurther active pharmaceutical ingredient. For example, an artificialnucleic acid as described herein encoding at least one polypeptide asdescribed herein may be co-administered in order to induce or enhance animmune response.

A further component of the inventive vaccine or composition may be animmunotherapeutic agent that can be selected from immunoglobulins,preferably IgGs, monoclonal or polyclonal antibodies, polyclonal serumor sera, etc., most preferably immunoglobulins directed against aHenipvirus and/or a Nipah virus and/or a Hendra virus. Preferably, sucha further immunotherapeutic agent may be provided as a peptide/proteinor may be encoded by a nucleic acid, preferably by a DNA or an RNA, morepreferably an mRNA. Such an immunotherapeutic agent allows providingpassive vaccination additional to active vaccination triggered by theinventive artificial nucleic acid or by the inventive polypeptide.

In a further aspect the invention provides a method of treating orpreventing a disorder, wherein the disorder is preferably an infectionwith Henipvirus and/or a Nipah virus and/or a Hendra virus or a disorderrelated to an infection with Henipvirus and/or a Nipah virus and/or aHendra virus, wherein the method comprises administering to a subject inneed thereof the artificial nucleic acid according to the invention, theinventive composition comprising at least one artificial nucleic acidaccording to the invention, the inventive polypeptides as describedherein, the inventive composition comprising at least one inventivepolypeptide, the inventive vaccine or the inventive kit or kit of parts.

In particular, such a method may preferably comprise the steps of:

-   a) providing the artificial nucleic acid according to the invention,    the inventive composition comprising at least one artificial nucleic    acid according to the invention, the inventive polypeptides as    described herein, the inventive composition comprising at least one    inventive polypeptide, the inventive vaccine or the inventive kit or    kit of parts;-   b) applying or administering the artificial nucleic acid according    to the invention, the inventive composition comprising at least one    artificial nucleic acid according to the invention, the inventive    polypeptides as described herein, the inventive composition    comprising at least one inventive polypeptide, the inventive vaccine    or the inventive kit or kit of parts to a tissue or an organism;-   c) optionally administering immunoglobuline (IgGs) against    Henipvirus and/or a Nipah virus and/or a Hendra virus.

According to a further aspect, the present invention also provides amethod for expression of at least one polypeptide comprising at leastone Henipvirus and/or a Nipah virus and/or a Hendra virus, or a fragmentor variant thereof, wherein the method preferably comprises thefollowing steps:

a) providing the inventive artificial nucleic acid comprising at leastone coding region encoding at least one polypeptide comprising at leastone Henipvirus and/or a Nipah virus and/or a Hendra virus, or a fragmentor variant thereof, preferably as defined herein, or a compositioncomprising said artificial nucleic acid; andb) applying or administering the inventive artificial nucleic acid orthe inventive composition comprising said artificial nucleic acid to anexpression system, e.g. to a cell-free expression system, a cell (e.g.an expression host cell or a somatic cell), a tissue or an organism.

The method may be applied for laboratory, for research, for diagnostic,for commercial production of peptides or proteins and/or for therapeuticpurposes. In this context, typically after preparing the inventiveartificial nucleic acid as defined herein or of the inventivecomposition or vaccine as defined herein, it is typically applied oradministered to a cell-free expression system, a cell (e.g. anexpression host cell or a somatic cell), a tissue or an organism, e.g.in naked or complexed form or as a (pharmaceutical) composition orvaccine as described herein, preferably via transfection or by using anyof the administration modes as described herein. The method may becarried out in vitro, in vivo or ex vivo. The method may furthermore becarried out in the context of the treatment of a specific disease,particularly in the treatment of infectious diseases, preferablyHenipvirus and/or a Nipah virus and/or a Hendra virus infection or arelated disorder as defined herein.

In this context, in vitro is defined herein as transfection ortransduction of the inventive artificial nucleic acid as defined hereinor of the inventive composition or vaccine as defined herein into cellsin culture outside of an organism; in vivo is defined herein astransfection or transduction of the inventive artificial nucleic acid orof the inventive composition or vaccine into cells by application of theinventive mRNA or of the inventive composition to the whole organism orindividual and ex vivo is defined herein as transfection or transductionof the inventive artificial nucleic acid or of the inventive compositionor vaccine into cells outside of an organism or individual andsubsequent application of the transfected cells to the organism orindividual.

Likewise, according to another aspect, the present invention alsoprovides the use of the inventive artificial nucleic acid as definedherein or of the inventive composition or vaccine as defined herein,preferably for diagnostic or therapeutic purposes, for expression of anencoded Henipvirus and/or a Nipah virus and/or a Hendra virus antigenicpeptide or protein, e.g. by applying or administering the inventiveartificial nucleic acid as defined herein or of the inventivecomposition or vaccine as defined herein, e.g. to a cell-free expressionsystem, a cell (e.g. an expression host cell or a somatic cell), atissue or an organism. The use may be applied for a (diagnostic)laboratory, for research, for diagnostics, for commercial production ofpeptides or proteins and/or for therapeutic purposes. In this context,typically after preparing the inventive artificial nucleic acid asdefined herein or of the inventive composition or vaccine as definedherein, it is typically applied or administered to a cell-freeexpression system, a cell (e.g. an expression host cell or a somaticcell), a tissue or an organism, preferably in naked form or complexedform, or as a (pharmaceutical) composition or vaccine as describedherein, preferably via transfection or by using any of theadministration modes as described herein. The use may be carried out invitro, in vivo or ex vivo. The use may furthermore be carried out in thecontext of the treatment of a specific disease, particularly in thetreatment of NIPAH virus infection or a related disorder.

In a particularly preferred embodiment, the invention provides theartificial nucleic acid, the inventive composition or the inventivevaccine for use as defined herein, preferably for use as a medicament,for use in treatment or prophylaxis, preferably treatment or prophylaxisof a Henipvirus and/or a Nipah virus and/or a Hendra virus infection ora related disorder, or for use as a vaccine.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 : shows that mRNA encoding Nipah virus F protein (R6311) inducesspecific humoral immune responses after immunization in mice. Furtherdetails are provided in Example 2.

FIG. 2 : shows that mRNA encoding Henipavirus G protein is expressed incells after transfection. Further details are provided in Example 3.

EXAMPLES

The Examples shown in the following are merely illustrative and shalldescribe the present invention in a further way. These Examples shallnot be construed to limit the present invention thereto.

Example 1: Preparation of mRNA Constructs for In Vitro and In VivoExperiments

For the present examples, DNA sequences encoding Nipah virus proteins aswell as DNA sequences encoding Hendra virus proteins are prepared andused for subsequent RNA in vitro transcription reactions. The generatedcoding sequences (RNA sequences) are provided in the sequence listing(SEQ ID NOs: 27-234, 599-806, 833-1040, 1067-1274, 1275-1508). DNAsequences are prepared by modifying the wild type encoding DNA sequencesby introducing a GC-optimized sequence for stabilization, using an insilico algorithms that increase the GC content of the respective codingsequence. Moreover, sequences are introduced into a pUC19 derived vectorand modified to comprise stabilizing sequences derived fromalpha-globin-3′-UTR, a stretch of 30 cytosines, a histone-stem-loopstructure, and a stretch of 64 adenosines at the 3′-terminal end(poly-A-tail) (indicated as “mRNA design 1” in Table 5, Table 6, Table7). Other sequences were introduced into a pUC19 derived vector tocomprise stabilizing sequences derived from 32L4 5′-UTR ribosomal 5′TOPUTR and 3′-UTR derived from albumin 7, a stretch of 30 cytosines, ahistone-stem-loop structure, and a stretch of 64 adenosines at the3′-terminal end (poly-A-tail) (indicated as “mRNA design 2” in Table 5,Table 6, Table 7). The obtained plasmid DNA constructs are transformedand propagated in bacteria (Escherichia coli) using common protocolsknown in the art.

RNA In Vitro Transcription on Linearized pDNA:

The DNA plasmids prepared according to paragraph 1 are enzymaticallylinearized using EcoRI and transcribed in vitro using DNA dependent T7RNA polymerase in the presence of a nucleotide mixture and cap analog(m7GpppG) under suitable buffer conditions. RNA production is performedunder current good manufacturing practice according to WO2016180430. Theobtained mRNAs are purified using PureMessenger® (CureVac, Tubingen,Germany; WO2008077592) and used for in vitro and in vivo experiments.

RNA In Vitro Transcription on PCR Amplified DNA Templates:

DNA plasmids prepared according to paragraph 1, or synthic DNAconstructs are used for PCR-amplification. The generated PCR templatesare used for subsequent RNA in vitro transcription using DNA dependentT7 RNA polymerase in the presence of a nucleotide mixture and cap analog(m7GpppG) under suitable buffer conditions. The obtained mRNA constructsare purified using PureMessenger® (CureVac, Tubingen, Germany;WO2008077592) and used for in vitro and in vivo experiments. Thegenerated mRNA constructs are indicated as “mRNA design 3” Table 5 andTable 6.

TABLE 7 mRNA constructs used in the Example section: SEQ ID NO: NameProtein mRNA NIPAV(Malaysia) 1 SEQ ID NO: 1353 mRNA design 2; opt1NIPAV(Malaysia) 12 SEQ ID NO: 1364 mRNA design 2; opt1NIPAV(Bangladesh2004) 3 SEQ ID NO: 1355 mRNA design 2; opt1NIPAV(Bangladesh2004) 13 SEQ ID NO: 1365 mRNA design 2; opt1HeV(Horse-Autralia-Hendra-1994)-F 8 SEQ ID NO: 1360 mRNA design 2; opt1HeV(Horse-Autralia-Hendra-1994)-G 19 SEQ ID NO: 1371 mRNA design 2; opt1IgE-leader(GC)_HeV(Horse-Autralia- 825 SEQ ID NO: 1397Hendra-1994)-G(71-604) mRNA design 2; opt1IgE-leader_Nipha(Bangladesh2004)-F 809 SEQ ID NO: 1381 mRNA design 2;opt1 SP-Influenza- 1043 SEQ ID NO: 1407 HA_Nipha(Bangladesh2004)-F mRNAdesign 2; opt1 SP-Osteonectin 1513 SEQ ID NO: 1543BM40_Nipha(Bangladesh2004)-F mRNA design 2; opt1 SP-HsChemo- 1514 SEQ IDNO: 1544 tripsinogen_Nipha(Bangladesh2004) mRNA design 2; opt1SP-Nipha(Malaysia1999)-F(1- 1515 SEQ ID NO: 154526)_Nipha(Bangladesh2004)-F(27-546) mRNA design 2; opt1

Example 2: Vaccination of Mice with mRNA Encoding Nipah

The results of the present Example shows that mRNA encoding Nipah virusF protein (NIV F Malaysia 1999; R6311) is expressed in mice afterintradermal injection. In addition, the expressed Nipah virus F proteinprovided by the inventive mRNA of the invention induces specific humoralimmune responses after immunization in mice.

Preparation of Protamine Complexed mRNA (“Vaccine Composition 1”)

Nipah virus mRNA construct (SEQ ID NO: 1353) was prepared as describedin Example 1 (RNA in vitro transcription). HPLC purified mRNA wascomplexed with protamine prior to use in in vivo vaccinationexperiments. The mRNA complexation consisted of a mixture of 50% freemRNA and 50% mRNA complexed with protamine at a weight ratio of 2:1.First, mRNA was complexed with protamine by addition ofprotamine-Ringer's lactate solution to mRNA. After incubation for 10minutes, when the complexes were stably generated, free mRNA was added,and the final concentration of the vaccine was adjusted with Ringer'slactate solution.

Immunization:

Female BALB/c mice were injected intradermally (i.d.) with mRNA vaccinecompositions with doses, application routes and vaccination schedules asindicated in Table A. As a negative control, one group of mice wasvaccinated with buffer (ringer lactate). All animals were vaccinated onday 0, 21 and 42. Blood samples were collected on day 21, 35, and 56 forthe determination of antibody titers.

TABLE A Vaccination regimen (Example 2): Number of mice Vaccinecomposition Dose Route/Volume 10 NIV F (Malaysia 1999) R6311; 80 μg i.d.2 × 50 μl Vaccine composition 1 10 100% RiLa Control i.d. 2 × 25 μl

Detection of Specific Humoral Immune Responses:

Hela cells were transfected with 2 μg of either R6311 vaccinecomposition using lipofectamine. The cells were harvested 20 h posttransfection, and seeded at 1×10⁵ per well into an 96 well plate. Thecells were incubated with sera of R6311 vaccinated mice (diluted 1:50)followed by aFITC-conjugated anti-mouse IgG antibody. Cells wereacquired on BD FACS Canto II using DIVA software and analyzed by FlowJo.

Results:

As shown in FIG. 1 , the mRNA encoding Nipah virus F protein (NIV FMalaysia 1999; R6311) is expressed in mice after i.d. administration.Moreover, as specific anti-NIV F IgGs were detected in sera of immunizedmice, the results also show that the applied mRNA vaccine is suitable toinduce specific humoral immune responses.

The results exemplify that the inventive mRNA-based Nipah virus vaccineworks and that similar mRNA vaccines comprising alternative mRNAconstructs according to the invention may also be suitably used.

Example 3: Expression Analysis of Nipah Virus and Hendra Virus GProteins Using Western Blot

The results of the present Example shows that mRNA encoding Nipah virusG protein and Hendra virus G protein are expressed in HeLa cells aftertransfection.

For the analysis of Nipah virus protein and Hendra virus G proteinexpression, HeLa cells were transfected with 2 μg unformulated mRNA (wfias negative control) using Lipofectamine as the transfection agent 20hours post transfection, HeLa cells were detached by trypsin-free/EDTAbuffer, harvested, and cell lysates were prepared. Cell lysates weresubjected to SDS-PAGE followed by western blot detection. Western Blotanalysis was performed using an anti-NIV G protein polyclonal IgG serumfraction (custom made by through immunization of rabbits with peptidesfrom NIV G (with x-reactivity to HeV G protein)) used in a 1:200dilution in combination with secondary anti rabbit antibody coupled toIRDye 800CW (Licor Biosciences). The presence of αβ-tubulin was analyzed(αβ-tubulin; Cell Signalling Technology; 1:1000 diluted) in combinationwith secondary anti rabbit antibody coupled to IRDye 680RD (LicorBiosciences). Inactivated Nipah virus was used as positive control forthe western blot (indicated as “ctr” in FIG. 2 ). The outline of theexperiment is shown in Table B. The result of the experiment is shown inFIG. 2 .

TABLE B Expression analysis experiment (Example 2): Lane SEQ ID NOTransfected composition 1 1364 Nipah virus G (Malaysia) R6003 2 1365Nipah virus G (Bangladesh) R6007 3 1371 Hendra virus G R6011 4 — wfi

Results:

As shown in FIG. 2 , the mRNA encoding Henipavirus G protein isexpressed in HeLa cells as the immunostaining for cell lysates of mRNAtransfected cells was substantially increased compared to the wficontrol group. In particular, immunostaining at about 70 kDa (G monomer)and about 260 kDa (G multimer) were detected. The results exemplify thatthe inventive mRNA encoding Henipavirus G protein is translated in cellsand that alternative mRNA constructs according to the invention may alsobe translated in cells, which is a prerequisite for an mRNA-basedvaccine.

Example 4: Expression of Nipah Virus and Hendra Virus Proteins in HeLaCells and Analysis by FACS

To determine in vitro protein expression of the constructs, HeLa cellsare transiently transfected with mRNA encoding Nipah virus (NiV) andHendra virus (HeV) antigens and stained using suitable customizedanti-NiV antibodies (raised in mouse) and anti-HeV antibodies,counterstained with a FITC-coupled secondary antibody (F5262 fromSigma). HeLa cells are seeded in a 6-well plate at a density of 400,000cells/well in cell culture medium (RPMI, 10% FCS, 1% L-Glutamine, 1%Pen/Strep), 24 h prior to transfection. HeLa cells are transfected with1 and 2 μg unformulated mRNA using Lipofectamine 2000 (Invitrogen). ThemRNA constructs according to Example 1 are used in the experiment,including a negative control encoding an irrelevant protein. 24 hourspost transfection, HeLa cells are stained with suitable anti anti-NiV oranti-HeV antibodies (raised in mouse; 1:500) and anti-mouse FITClabelled secondary antibody (1:500) and subsequently analyzed by flowcytometry (FACS) on a BD FACS Canto II using the FACS Diva software.Quantitative analysis of the fluorescent FITC signal is performed usingthe FlowJo software package (Tree Star, Inc.).

Example 5: Analysis of Expression and Secretion of Nipah Virus andHendra Virus Proteins Using Western Blot

For the analysis of Nipah virus protein and Hendra virus proteinsecretion, HeLa cells are transfected with 1 μg and 2 μg unformulatedmRNA (including a negative control encoding an irrelevant protein) usingLipofectamine as the transfection agent. Supernatants, harvested 24hours post transfection, are filtered through a 0.2 μm filter. Clarifiedsupernatants are applied on top of 1 ml 20% sucrose cushion (in PBS) andcentrifuged at 14000 rcf (relative centrifugal force) for 2 hours at 4°C. Protein content is analyzed by Western Blot using anti-NiV andanti-HeV antibodies as primary antibody in combination with secondaryanti mouse antibody coupled to IRDye 800CW (Licor Biosciences). Thepresence of αβ-tubulin is also analyzed as control for cellularcontamination (αβ-tubulin; Cell Signalling Technology; 1:1000 diluted)in combination with secondary anti rabbit antibody coupled to IRDye680RD (Licor Biosciences). For the analysis of NiV and HeV proteins incell lysates, HeLa cells are transfected with 1 μg and 2 μg unformulatedmRNAs (generated according to Example 1) including a negative controlencoding an irrelevant protein using Lipofectamine as the transfectionagent 24 hours post transfection, HeLa cells are detached bytrypsin-free/EDTA buffer, harvested, and cell lysates are prepared. Celllysates are subjected to SDS-PAGE under non-denaturating/non-reductingfollowed by western blot detection. Western Blot analysis is performedusing a anti NiV and anti-HeV antibodies as primary antibody incombination with secondary anti mouse antibody coupled to IRDye 800CW(Licor Biosciences).

Example 6: Preparation of Nipah Virus and Hendra Virus VaccineCompositions

For further in vivo vaccination experiments, different compositions ofNipah virus mRNA vaccine and Hendra virus mRNA vaccine are preparedusing constructs obtained in Example 1. One composition comprisesprotamine-complexed mRNA, one composition comprises mRNA that isformulated without protamine (“naked”), one composition comprises mRNAthat is encapsulated in lipid nanoparticles (LNPs), and one compositioncomprises polymer-lipidoid complexed mRNA.

Nipah virus and Hendra virus mRNA constructs are complexed as describedin Example 2.

Nipah virus and Hendra virus mRNA constructs are formulated withoutprotamine. The final concentration of the vaccine is adjusted withRingers lactate solution.

Preparation of LNP Encapsulated mRNA (“Vaccine Composition 3”)

A lipid nanoparticle (LNP)-encapsulated mRNA mixture is prepared usingan ionizable amino lipid (cationic lipid), phospholipid, cholesterol anda PEGylated lipid. LNPs are prepared as follows. Cationic lipid, DSPC,cholesterol and PEG-lipid are solubilized in ethanol. Briefly, mRNAmixture is diluted to a total concentration of 0.05 mg/mL in 50 mMcitrate buffer, pH 4. Syringe pumps are used to mix the ethanolic lipidsolution with the mRNA mixture at a ratio of about 1:6 to 1:2 (vol/vol).The ethanol is then removed and the external buffer replaced with PBS bydialysis. Finally, the lipid nanoparticles are filtered through a 0.2 μmpore sterile filter. Lipid nanoparticle particle diameter size isdetermined by quasi-elastic light scattering using a Malvern ZetasizerNano (Malvern, UK).

Preparation of Polymer-Lipidoid Complexed mRNA (“Vaccine Composition 4”)

20 mg peptide (CHHHHHHRRRRHHHHHHC—NH2; SEQ ID NO: 309) TFA salt isdissolved in 2 mL borate buffer pH 8.5 and stirred at room temperaturefor approximately 18 h. Then, 12.6 mg PEG-SH 5000 (Sunbright) dissolvedin N-methylpyrrolidone is added to the peptide solution and filled up to3 mL with borate buffer pH 8.5. After 18 h incubation at roomtemperature, the reaction mixture is purified and concentrated bycentricon procedure (MWCO 10 kDa), washed against water, andlyophilized. The obtained lyophilisate is dissolved in ELGA water andthe concentration of the polymer is adjusted to 10 mg/mL. The obtainedpolyethylene glycol/peptide polymers (HO-PEG5000-S—(S—CHHHHHHRRRRHHHHHHC—S-)7-S-PEG 5000-OH-amino acid component:SEQ ID NO: 309) are used for further formulation and are hereinafterreferred to as PB83.

Preparation of 3-C12-OH Lipidoid

First, lipidoid 3-C12 was obtained by acylation oftris(2-aminoethyl)amine with an activated lauric (C12) acid derivative,followed by reduction of the amide. Alternatively, it may be prepared byreductive amination with the corresponding aldehyde. Lipidoid 3-C12-OHwas prepared by addition of the terminal C12 alkyl epoxide with the sameoligoamine according to Love et al., pp. 1864-1869, PNAS, vol. 107(2010), no. 5.

Preparation of Compositions with Nanoparticles of Polymer-LipidoidComplexed mRNA

First, ringer lactate buffer (RiLa; alternatively e.g. saline (NaCl) orPBS buffer may be used), respective amounts of lipidoid, and respectiveamounts of a polymer (PB83) are mixed to prepare compositions comprisinga lipidoid and a peptide or polymer. Then, the carrier compositions areused to assemble nanoparticles with the mRNA by mixing the mRNA withrespective amounts of polymer-lipidoid carrier and allowing anincubation period of 10 minutes at room temperature such as to enablethe formation of a complex between the lipidoid, polymer and mRNA. Inorder to characterize the integrity of the obtained polymer-lipidoidcomplexed mRNA particles, RNA agarose gel shift assays are performed. Inaddition, size measurements are performed (gel shift assay, Zetasizer)to evaluate whether the obtained nanoparticles have a uniform sizeprofile.

Example 7: Vaccination of Mice and Evaluation of Nipah Virus SpecificImmune Response

Female BALB/c mice are injected intradermally (i.d.) and intramuscularly(i.m.) with respective mRNA vaccine compositions (prepared according toExample 6) with doses, application routes and vaccination schedules asindicated in Table C. As a negative control, one group of mice isvaccinated with buffer (ringer lactate). All animals are vaccinated onday 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 forthe determination of binding and neutralizing antibody titers (seebelow).

TABLE C Vaccination regimen - Nipah virus experiment (Example 7) Numberof Route/ Vaccination Group mice Vaccine composition Volume Schedule(day) 1 10 40 μg Nipah virus i.d. 0/21/35 RNA Composition 1 2 × 25 μl 210 40 μg Nipah virus i.m. 0/21/35 RNA Composition 1 2 × 25 μl 3 10 20 μgNipah virus i.d. 0/21/35 RNA Composition 2 2 × 25 μl 4 10 20 μg Nipahvirus i.m. 0/21/35 RNA Composition 2 2 × 25 μl 5 10 10 μg Nipah virusi.d. 0/21/35 RNA Composition 3 2 × 25 μl 6 10 10 μg Nipah virus i.m.0/21/35 RNA Composition 3 2 × 25 μl 7 10 100% RiLa Control i.d. 0/21/352 × 25 μl

Determination of Anti Nipah Virus Protein Antibodies by ELISA:

ELISA is performed using inactivated Nipah virus infected cell lysatefor coating. Coated plates are incubated using respective serumdilutions, and binding of specific antibodies to the Nipah virusantigens are detected using biotinylated isotype specific anti-mouseantibodies followed by streptavidin-HRP (horse radish peroxidase) withABTS as substrate. Endpoint titers of antibodies directed against theNipah virus antigens are measured by ELISA on day 63 after threevaccinations.

Intracellular Cytokine Staining:

Splenocytes from vaccinated mice are isolated according to a standardprotocol known in the art. Briefly, isolated spleens are grinded througha cell strainer and washed in PBS/1% FBS followed by red blood celllysis. After an extensive washing step with PBS/1% FBS splenocytes areseeded into 96-well plates (2×10⁶ cells per well). The cells arestimulated with a mixture of four Nipah virus protein specific peptideepitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of ananti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in thepresence of a protein transport inhibitor. After stimulation, cells arewashed and stained for intracellular cytokines using theCytofix/Cytoperm reagent (BD Biosciences) according to themanufacturer's instructions. The following antibodies are used forstaining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC(1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) andincubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguishlive/dead cells (Invitrogen). Cells are acquired using a Canto II flowcytometer (Beckton Dickinson). Flow cytometry data is analyzed usingFlowJo software package (Tree Star, Inc.)

Nipah Virus Plaque Reduction Neutralization Test (PRNT50):

Sera are analyzed by a plaque reduction neutralization test (PRNT50),performed as commonly known in the art. Briefly, obtained serum samplesof vaccinated mice are incubated with Nipah virus. That mixture is usedto infect cultured cells, and the reduction in the number of plaques isdetermined.

Example 8: Vaccination of Mice and Evaluation of Hendra Virus SpecificImmune Response

Female BALB/c mice are injected intradermally (i.d.) and intramuscularly(i.m.) with respective mRNA vaccine compositions (prepared according toExample 6) with doses, application routes and vaccination schedules asindicated in Table D. As a negative control, one group of mice isvaccinated with buffer (ringer lactate). All animals are vaccinated onday 1, 21 and 35. Blood samples are collected on day 21, 35, and 63 forthe determination of binding and neutralizing antibody titers (seebelow).

Determination of Anti Hendra Virus Protein Antibodies by ELISA:

ELISA is performed using inactivated Hendra virus infected cell lysatefor coating. Coated plates are incubated using respective serumdilutions, and binding of specific antibodies to the Hendra virusantigens are detected using biotinylated isotype specific anti-mouseantibodies followed by streptavidin-HRP (horse radish peroxidase) withABTS as substrate. Endpoint titers of antibodies directed against theHendra virus antigens are measured by ELISA on day 63 after threevaccinations.

TABLE D Vaccination regimen - Hendra virus experiment (Example 8):Number of Route/ Vaccination Group mice Vaccine composition VolumeSchedule (day) 1 10 40 μg Hendra virus i.d. 0/21/35 RNA Composition 1 2× 25 μl 2 10 40 μg Hendra virus i.m. 0/21/35 RNA Composition 1 2 × 25 μl3 10 20 μg Hendra virus i.d. 0/21/35 RNA Composition 2 2 × 25 μl 4 10 20μg Hendra virus i.m. 0/21/35 RNA Composition 2 2 × 25 μl 5 10 10 μgHendra virus i.d. 0/21/35 RNA Composition 3 2 × 25 μl 6 10 10 μg Hendravirus i.m. 0/21/35 RNA Composition 3 2 × 25 μl 7 10 100% RiLa Controli.d. 0/21/35 2 × 25 μl

Intracellular Cytokine Staining:

Splenocytes from vaccinated mice are isolated according to a standardprotocol known in the art. Briefly, isolated spleens are grinded througha cell strainer and washed in PBS/1% FBS followed by red blood celllysis. After an extensive washing step with PBS/1% FBS splenocytes areseeded into 96-well plates (2×10⁶ cells per well). The cells arestimulated with a mixture of four Hendra virus protein specific peptideepitopes (5 μg/ml of each peptide) in the presence of 2.5 μg/ml of ananti-CD28 antibody (BD Biosciences) for 6 hours at 37° C. in thepresence of a protein transport inhibitor. After stimulation, cells arewashed and stained for intracellular cytokines using theCytofix/Cytoperm reagent (BD Biosciences) according to themanufacturer's instructions. The following antibodies are used forstaining: CD3-FITC (1:100), CD8-PE-Cy7 (1:200), TNF-PE (1:100), IFNγ-APC(1:100) (eBioscience), CD4-BD Horizon V450 (1:200) (BD Biosciences) andincubated with Fcγ-block diluted 1:100. Aqua Dye is used to distinguishlive/dead cells (Invitrogen). Cells are acquired using a Canto II flowcytometer (Beckton Dickinson). Flow cytometry data is analyzed usingFlowJo software package (Tree Star, Inc.)

Nipah Virus Plaque Reduction Neutralization Test (PRNT50):

Sera are analyzed by a plaque reduction neutralization test (PRNT50),performed as commonly known in the art. Briefly, obtained serum samplesof vaccinated mice are incubated with Hendra virus. That mixture is usedto infect cultured cells, and the reduction in the number of plaques isdetermined.

Example 9: Vaccination of Mice Using Polymer-Lipidoid Complexed RNA andEvaluation of Immune Response

Female BALB/c mice are injected intramuscularly (i.m.) with respectivemRNA vaccine compositions (prepared according to Example 6) with doses,application routes and vaccination schedules as indicated in Table F. Asa negative control, one group of mice is vaccinated with buffer (ringerlactate). All animals are vaccinated on day 1, 21 and 35. Blood samplesare collected on day 21, 35, and 63 for the determination of binding andneutralizing antibody titers (see below).

Evaluation of Specific Immune Responses:

ELISA is performed using inactivated Hendra virus or Nipah virusinfected cell lysate for coating (as described above). Splenocytes fromvaccinated mice are isolated according to a standard protocol known inthe art and intracellular cytokine staining is performed as describedabove. In addition, sera are analyzed by a plaque reductionneutralization test (PRNT50), performed as described above.

TABLE F Vaccination regimen (Example 9): Number of Route/ VaccinationGroup mice Vaccine composition Volume Schedule (day) 1 10 40 μg Hendravirus i.m. 0/21/35 RNA Composition 4 2 × 25 μl 2 10 40 μg Nipah virusi.m. 0/21/35 RNA Composition 4 2 × 25 μl 3 10 100% RiLa Control i.d.0/21/35 2 × 25 μl

Example 10: Clinical Development of a Nipah Virus and Hendra Virus mRNAVaccine Composition

To demonstrate safety and efficiency of the Nipah virus and Hendra virusmRNA vaccine composition, a clinical trial (phase I) is initiated. Inthe clinical trial, a cohort of human volunteers is intradermally orintramuscularly injected for at least two times. In order to assess thesafety profile of the vaccine compositions according to the invention,subjects are monitored after administration (vital signs, vaccinationsite tolerability assessments, hematologic analysis). The efficacy ofthe immunization is analyzed by determination of virus neutralizingtiters (VNT) in sera from vaccinated subjects. Blood samples arecollected on day 0 as baseline and after completed vaccination. Sera areanalyzed for virus neutralizing antibodies.

The invention claimed is:
 1. A method of treating or preventing adisorder, wherein the method comprises applying or administering to asubject in need thereof an effective amount of a composition comprisinga RNA comprising at least one coding sequence encoding a Nipah virusfusion protein F: (a) at least about 95% identical to the sequence ofSEQ ID NO: 1 and wherein the Nipah virus fusion protein F is encoded bya RNA coding sequence at least about 95% identical to SEQ ID NO: 53; or(b) at least about 95% identical to the sequence of SEQ ID NO: 573 andwherein the Nipah virus fusion protein F is encoded by a RNA codingsequence at least about 95% identical to SEQ ID NO:
 625. 2. The methodaccording to claim 1, wherein the at least one coding sequence encodes aNipah virus fusion protein F at least about 95% identical to thesequence of SEQ ID NO:
 573. 3. The method according to claim 2, whereinthe at least one coding sequence encodes a Nipah virus fusion protein Fidentical to the sequence of SEQ ID NO:
 573. 4. The method according toclaim 1, wherein the RNA encodes a Nipah virus fusion protein Fidentical or at least 95% identical to SEQ ID NO:
 1. 5. The methodaccording to claim 1, wherein the RNA encodes at least one furtherprotein element selected from a secretory signal peptide, atransmembrane domain, a VLP forming domain, a peptide linker, aself-cleaving peptide, an immunologic adjuvant sequence, and/or adendritic cell targeting sequence.
 6. The method according to claim 1,wherein the RNA is bicistronic or multicistronic.
 7. The methodaccording to claim 1, wherein the RNA is a mRNA.
 8. The method accordingto claim 1, wherein the G/C content of the at least one coding sequenceis increased compared to the G/C content of the corresponding wild typecoding sequence, and/or wherein the C content of the at least one codingsequence is increased compared to the C content of the correspondingwild type coding sequence and/or wherein the codons in the at least onecoding sequence are adapted to human codon usage, wherein the codonadaptation index (CAI) is increased or maximised in the at least onecoding sequence, wherein the amino acid sequence encoded by the at leastone coding sequence is not modified compared to the amino acid sequenceencoded by the corresponding wild type coding sequence.
 9. The methodaccording to claim 1, wherein the RNA comprises an untranslated region(UTR).
 10. The method according to claim 1, comprising a plurality ofRNA molecules encoding different antigenic protein.
 11. The methodaccording to claim 10, wherein (i) each of the RNA molecules encodes adifferent antigenic protein derived from a Henipavirus and/or Hendravirus and/or Nipah virus; (ii) each of the RNA molecules encodes adifferent antigenic protein derived from genetically the sameHenipavirus and/or Hendra virus and/or Nipah virus; or (iii) each of theRNA molecules encodes a different antigenic protein derived from agenetically different Henipavirus and/or Hendra virus and/or Nipahvirus.
 12. The method according to claim 1, wherein RNA is complexedwith one or more cationic or polycationic component.
 13. The methodaccording to claim 1, wherein the artificial nucleic acid is complexedwith one or more polysaccharides.
 14. The method according to claim 1,wherein the RNA is complexed with one or more lipids, thereby formingliposomes, lipid nanoparticles and/or lipoplexes.
 15. The methodaccording to claim 1, wherein the composition comprises at least oneadjuvant component.
 16. The method according to claim 1, wherein the RNAcomprises in 5′ to 3′ direction, the following elements a)-g): a) 5′-capstructure; b) optionally, 5′-UTR element; c) at least one codingsequence encoding the Nipah virus fusion protein F; d) a 3′-UTR element;e) optionally, poly(A) sequence; f) optionally, poly(C) sequence; and g)optionally, a histone stem-loop.
 17. The method according to claim 16,wherein the RNA comprises, in the 5′ to 3′ direction, the followingelements a)-g): a) 5′-cap structure; b) a 5′-UTR element; c) the atleast one coding sequence encoding the Nipah virus fusion protein F; d)a 3′-UTR element; e) a poly(A) sequence; f) optionally, poly(C)sequence; and g) optionally, a histone stem-loop.
 18. The methodaccording to claim 12, wherein the cationic or polycationic component isa cationic or polycationic polymer, a cationic or polycationic peptideor protein, or a cationic or polycationic lipid.
 19. The methodaccording to claim 12, wherein the cationic or polycationic peptide orprotein is a protamine.